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Role of CrmA in regulating inflammation and apoptosis during cowpox virus infection

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Title:
Role of CrmA in regulating inflammation and apoptosis during cowpox virus infection
Creator:
Nathaniel, Rajkumar, 1971-
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English
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xvi, 239 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Apoptosis ( jstor )
Chickens ( jstor )
Cytokines ( jstor )
Enzymes ( jstor )
Infections ( jstor )
Inflammation ( jstor )
Molecules ( jstor )
Receptors ( jstor )
Serpins ( jstor )
Viruses ( jstor )
Apoptosis ( mesh )
Capases ( mesh )
Chick Embryo ( mesh )
Cowpox ( mesh )
Department of Molecular Genetics and Microbiology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Molecular Genetics and Microbiology -- UF ( mesh )
Immunity ( mesh )
Inflammation ( mesh )
Interleukins ( mesh )
Poxviridae Infections ( mesh )
Research ( mesh )
Serpins ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references (leaves 196-238).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Nathaniel Rajkuma.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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70171164 ( OCLC )

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ROLE OF CrmA IN REGULATING INFLAMMATION AND APOPTOSIS DURING
COWPOX VIRUS INFECTION














By

RAJKUMAR NATHANIEL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2003















ACKNOWLEDGMENTS

A number of people have contributed to the completion of this work. I would like

to thank my mentor Richard Moyer for allowing me to start this project and also for

helping me complete it. Dick has been extremely patient and forgiving with me these

past 6 and some years. He continuously made efforts to train me as a scientist. Dick

helped me appreciate poxviruses. The members of my committee Lung-Ji Chang, Paul

Gulig, and Michael Clare-Salzler have also been instrumental in guiding me through

these years. They have sat patiently through many more committee meetings than they

would have wanted to. I would also like to thank Dr. Richard Condit and Dr. Sue Moyer

for valuable input and suggestions during joint lab-meetings.

The members of the Moyer Lab need special mention. They have been good

friends and work colleagues. Mike Duke taught me the difference between Republicans

and Democrats. Mike and I have had many philosophical discussions about the world we

live in and I will miss those interactions. Alison Bawden, Traci Ness and Kristin Moon of

the so-called Estrogen Sanctuary made sure I wasn't naive by the time they left the lab.

Ben Luttge and Lauren Brum have been wonderful work mates and strongly encouraged

my graduation process. Peter Turner has been generous with his time and was always

there when I needed technical advice. I am grateful to Marie Becker, for reading my

manuscripts and for her mothering us in the lab. Qianjun Li has been a good friend, work

neighbor and advisor.









I am grateful to Joyce Conners for taking care of all the necessary paperwork. I

would also like to thank the administrative staff of the Department of Molecular Genetics

and Microbiology for their timely help.

I owe a lot to friends at the Graduate Evangelical Fellowship, who have seen me

through my graduate career. I would specially like to mention Chip Appel, whose

constant encouragement and friendship was invaluable at times. Brian and Shigeko

Jackson, Diomy Zamora, Kwabena Amphosa-Managera, Shelly Merves and Ricardo

Gomez have specially seen me through the final stages of completing this project. Their

friendship has been invaluable. My family, have patiently waited for me to finish this

degree and have been a positive influence through all these years. Finally, I would like to

express my heartfelt gratitude to my wife, Leena, for her love, patience and endurance

despite all the strain we have gone through my graduate career. Leena has truly been my

helpmate. I firmly believe in the notion that it takes a village to raise an individual.

There are many others, who have, in little ways influenced my life and shaped the way I

think. Like little drops of water that make a flowing river, these individuals have touched

my life in numerous ways.














TABLE OF CONTENTS
page

ACKNOW LEDGM ENTS .................................................................................................. ii

LIST OF TABLES........................................................................................................... viii

LIST OF FIGURES ........................................................................................................... ix

ABBREVIATION S ........................................................................................................... xi

ABSTRACT...................................................................................................................... xv

CHAPTER

1 LITERATURE REVIEW ............................................................................................... 1

Introduction..................................................................................................................... 1
Imm une System s............................................................................................................. 1
Innate Imm unity and Inflamm ation................................................................................ 3
Innate Imm une Recognition....................................................................................... 5
Toll and Interleukin-1 Receptors .................................................................................... 6
Toll and Toll Like Receptors (TLR).......................................................................... 6
Interleukin-1 (IL-1).................................................................................................... 8
Inflamm atory M editors ............................................................................................... 17
Tum or Necrosis Factor (TNF) ................................................................................. 17
Interleukin- 18 (IL- 18).............................................................................................. 21
Interleukin-8 (IL-8).................................................................................................. 25
Avian Cytokines............................................................................................................ 29
TNF-ax ...................................................................................................................... 29
IL-1 .......................................................................................................................... 30
IL-18 ........................................................................................................................ 32
CXC Chem okines .................................................................................................... 32
Viral Inhibitors of Inflamm ation................................................................................... 33
TNF Inhibitors ......................................................................................................... 33
IL-1 Inhibitors.......................................................................................................... 34
IL-18 Inhibitors........................................................................................................ 35
Chem okine Inhibitors............................................................................................... 36
Inhibitors of Complement........................................................................................ 38
Inhibitors of Interferons ........................................................................................... 39
Apoptosis ...................................................................................................................... 40









Caspases................................................................................................................... 44
Inflam m atory Caspases-1, -4, -5, -11 and -12.......................................................... 46
Apoptotic Initiator Caspases-2, -8, -9, and -10........................................................ 48
Apoptotic Effector Caspases-3, -6 and -7................................................................ 49
Intrinsic Activation of Caspases: Mitochondrial Permeability Transition Pore...... 51
Extrinsic Activation of Caspases: Death Receptor Mediated Signaling.................. 53
Granzym e B ............................................................................................................. 56
Apoptosis in Avian Species .......................................................................................... 57
Viral Inhibitors of Apoptosis ........................................................................................ 58
Viral Inhibitors of Mitochondria: PT Pore Modulators and Bcl-2 Homologs......... 59
vLIPs: Inhibitors of DISC ........................................................................................ 60
Baculovirus Caspase Inhibitor: P35......................................................................... 61
Baculovirus Inhibitor of Apoptosis Protein (IA P)................................................... 62
Caspase Independent Induction of Apoptosis............................................................... 64
Poxviruses..................................................................................................................... 65
Poxvirus Lifecycle ................................................................................................... 67
Im portance of Poxviruses......................................................................................... 69
Serine Protease Inhibitors (Serpins).............................................................................. 70
Poxvirus Serpins ........................................................................................................... 75
SERP2...................................................................................................................... 76
Cytokine Response M odifier A (Crm A).................................................................. 77
SPI-2 is a Crm A Hom ologue................................................................................... 80
RPV SPI-2 and CPV Crm A Equivalency..................................................................... 81
In Vivo Role of CrmA during CPV Infections of CAMs.............................................. 84

2 M ATERIA LS AN D M ETH OD S.................................................................................. 87

Virology ........................................................................................................................ 87
Cells ......................................................................................................................... 87
Viruses ..................................................................................................................... 87
Viral Stock Preparation and Quantification............................................................. 88
M olecular Techniques................................................................................................... 89
Polym erase Chain Reaction (PCR).......................................................................... 89
DNA Manipulation, Ligation and Transformation.................................................. 91
V iral DN A Preparation ............................................................................................ 92
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)...... 92
Im m unoblot.............................................................................................................. 93
Pulse Labeling.......................................................................................................... 94
DN A Sequencing ..................................................................................................... 94
Sequence Analysis and D database Search................................................................. 95
Recom binant V irus Construction.................................................................................. 95
Transfection of Infected Cells U sing Liposom es..................................................... 95
Selection of Recombinant Virus in the Presence of Mycophenolic Acid ................ 96
Reverse Selection of Recombinant Virus using 6-Thioguanine.............................. 99
Construction of Recombinant RPV Expressing CrmA............................................ 99
Construction of Recombinant RPV Expressing P35 ............................................. 100



v









Construction of Recombinant CPV Expressing SPI-2........................................... 102
Construction of Recombinant CPV Expressing P35 ............................................. 103
Construction of Recombinant CPV Expressing SERP2........................................ 104
Site Directed Mutagenesis of CrmA Gene............................................................. 104
Construction of CPVCrmA D303A....................................................................... 105
Construction of CPVAIL- 1 PR Virus ..................................................................... 105
A poptosis A ssays........................................................................................................ 106
DAPI Staining of Infected Cells........................................................................... 106
Preparation of Infected Cell Extracts for Caspase Activity Assay........................ 109
Ac-DEVD-AMC Cleavage Assay for Caspase Activity ....................................... 109
In Vivo Infection Assays using CAMs........................................................................ 110
Preparation of Chicken Chorioallantoic Membrane for Infections........................ 110
Infecting and Harvesting CAMs............................................................................ 111
Measurement of Reactive Oxygen Intermediates.................................................. I11
M TT R education A ssay........................................................................................... 112
V irus Infectivity A ssay .......................................................................................... 112
Terminal Caspase Activity Assay on Extracts from Infected CAMs.................... 113
Construction of Plasmids Containing Chicken ProlL- 11P or ProlL- 18.................. 114
Plasmid Containing Mouse ProlL- 1 3 .................................................................... 115
Quick Coupled Transcription/Translation System................................................. 115
In Vitro Cleavage Assay for Processing ProlL- 1 3 and ProlL- 18.......................... 115

3 R E SU LT S ................................................................................................................... 117

Equivalency of SPI-2 and CrmA................................................................................ 117
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Recombinant Virus Construction..... 118
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Prevent Apoptosis in Pig Cells........ 119
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Inhibit Caspase Activation............... 124
Recombinant RPV and CPV Viruses Expressing P35........................................... 130
RPVASPI-2::P35 Induces Apoptosis in LLC-PK1 Cells....................................... 131
CPVAcrmA::P35 Blocks Apoptotic Induction in LLC-PK1 Cells........................ 132
Viral Protein Expression: Effect of AraC.............................................................. 134
Recombinant CPV Viruses Expressing SERP2 or CrmA D303A.............................. 137
Infections of LLC-PK1 Cells with CPV Recombinants: DAPI Stained Cells....... 138
Infections of LLC-PK1 Cells with CPV Recombinants: Ac-DEVD-AMC
C leavage A activity .............................................................................................. 141
Recombinant CPV Infections of CAMs ..................................................................... 144
Pock Morphology of Recombinant CPV Infected CAMs..................................... 145
All White Pocks Contain Heterophils.................................................................... 145
Inflammatory Pocks Show High Levels ofNADPH Oxidase............................... 148
P35 and Serp2 Restore Viral Yields from Infected CAMs.................................... 152
P35, Serp2 and CrmA Block Induction of Terminal Caspase Activity within
Infected Pocks................................................................................................... 153
Effect of Caspase Inhibitors on Protease Activity Present in
Infected C A M s................................................................................................. 155
Chicken ProIL-13 Processing Activity in CAMs.................................................. 159









Processing ofProIL-1 p can be Blocked by either SERP2 or P35 within
Inflam m atory Pocks.......................................................................................... 162
Processing ofProlL-18 can be Blocked by either SERP2 or P35 within
Inflam m atory Pocks.......................................................................................... 164
Chicken ProlL- 18 Processing Activity in CPVAcrmA Lysates is Blocked by
Caspase Specific Peptide Inhibitors.................................................................. 166
Caspase-3 Activity in the Presence of Chicken CAM Extracts........................ 171
CPVAIL- 1 PR Fails to Induce Inflammation on CAMs.............................................. 171

4 D ISC U SSIO N ............................................................................................................. 178

Equivalency of SPI-2 and CrmA ................................................................................ 178
Potential A pplications............................................................................................ 181
P35 Replacements of SPI-2 and CrmA....................................................................... 183
Function of CrmA during CPV Infections of CAMs.................................................. 184
Requirement of Intact RCL for CrmA Function In Vivo....................................... 185
Regulation of Inflammation and Apoptosis by CrmA are Distinct Functions....... 185
Inflammation during CPV Infection may not be due to IL- 1 P or IL-18................ 186
Models of Inflammatory Response on CAMs....................................................... 190
Future Studies .............................................................................................. ............... 193

LIST OF REFERENCES................................................................................................ 196

BIOGRAPHICAL SKETCH.......................................................................................... 239


LLI vii














LIST OF TABLES
Table page

1 Characterization of mice deficient for different components of the IL-1 system......... 15
2 List of mice deficient in the IL-1 system ...................................................................... 16
3 Taxonomy of poxviruses............................................................................................... 66
4 List of Primers................................................................................... ............................ 90
5 List of plasmids constructed....................................................................................... 107
6 List of constructed recombinant viruses..................................................................... 108
7 Summary of results using recombinant CPVs............................................................ 177














LIST OF FIGURES
Figure page

1 Signaling by TNF, IL-1R and TLR........................................................................... 11
2 A poptosis .................................................................................................................. 43
3 Poxvirus replication cycle......................................................................................... 68
4 Serpins as physiological regulators........................................................................... 72
5 Serpin R C L ............................................................................................................... 73
6 Inhibition of trypsin by alpha-1 antitrypsin.............................................................. 74
7 Comparison of SPI-2 and CrmA peptide sequences................................................. 83
8 Construction of recombinant RPV............................................................................ 120
9 Construction of recom binant CPV ............................................................................ 121
10 Expression of SPI-2 and CrmA during RPV infections........................................... 122
11 Expression of CrmA and SPI-2 during CPV infections........................................... 123
12 Morphological characteristics of LLC-PK1 cells infected with RPV
derivatives ...................................................................................................... 125
13 Morphological characteristics of LLC-PKI cells infected with CPV
derivatives ...................................................................................................... 126
14 Biochemical changes in LLC-PK1 cells infected by RPV derivatives.................... 128
15 Biochemical changes in LLC-PK1 cells infected by CPV derivatives.................... 129
16 Comparison of P35 expressed in RPV and CPV..................................................... 133
17 Effect of AraC on P35 expression in recombinant RPV and CPV.......................... 136
18 Expression of SERP2 and CrmA D303A recombinant proteins ............................. 139
19 Morphological changes in LLC-PK1 cells infected with CPVAcrmA::SERP2
or CPV Crm A D 303A ..................................................................................... 142
20 Biochemical changes in LLC-PK1 cells infected with CPVAcrmA::SERP2
or CPV Crm A D 303A ..................................................................................... 143
21 Pock morphology on CAMs infected with recombinant viruses............................. 146
22 Histological examination of pocks from infected CAMs........................................ 149
23 Inflammatory pocks show similar levels of oxidative burst.................................... 151
24 P35 and SERP2 restore viral yields from infected CAMs....................................... 154
25 P35, SERP2 and CrmA block terminal caspase activity within CEF cells.............. 156
26 P35, SERP2 and CrmA block terminal caspase activity within infected pocks...... 157
27 Chicken IL-1 3 processing........................................................................................ 161
28 P35 and SERP2 function like CrmA to prevent Caspase-1 mediated
processing of prolL- I P3.................................................................................. 163
29 P35 and SERP2 function like CrmA to prevent processing of chicken
prolL-18 in C A M s ....................................................................................... 165
30 Caspase-1 mediated processing of chicken prolL-18 is blocked by peptides.......... 168
31 Chicken prolL-18 processing activity in CPVAcrmA::lacZ extracts is
inhibited by caspase specific peptides.......................................................... 169









32 Caspase-3 activity in the presence of chicken extracts............................................ 172
33 Construction of CPVAIL-13R................................................................................. 174
34 CPVAIL-1OR fails to induce inflammation during CAM infections ....................... 175














ABBREVIATIONS


AcNPV Autographa californica nucleopolyhedrovirus

AP-1 activator protein- 1

APC antigen presenting cell

BIR baculovirus IAP repeat domain

BV baculovirus

CAM chicken chorioallantoic membrane

CEF chicken embryo fibroblast

CpGV Cydiapomonella granulosisvirus

CPV cowpoxvirus

CrmA cytokine response modifier A

CTL cytotoxic T lymphocytes

CV-1 monkey kidney cell line

DD death domain

DFF DNA fragmentation factor

DNA-PK DNA protein kinase

DISC death receptor induced signaling complex

E.coli gpt E.coli guanine phosphoribosyltransferase

FADD Fas associated death domain

G-CSF granulocyte colony stimulating factor









GM-CSF granulocyte-monocyte colony stimulating factor

HPRT hypoxanthine guanine phosphoribosyltransferase

lAP inhibitors of apoptosis proteins from baculovirus

ICE interleukin 1 1 converting enzyme

IFN interferon

IL interleukin

IRAK IL- 1 R associated kinase

ITA inhibitor of T-cell apoptosis

ITR inverted terminal repeat

JNK Jun N-terminal kinase

kbp kilobase pairs

LLC-PK1 pig kidney cell line

MAPK mitogen activated protein kinase

M-CSF monocyte colony stimulating factor

MMP matrix metalloproteinase

MOI multiplicity of infection

MPA mycophenolic acid

NADP nicotinamide adenine dinucleotide phosphate

NF-KB nuclear factor kappa B

NGF nerve growth factor

NIK NF-KB inducing kinase

NK natural killer cell

NO nitric oxide









OpNPV Orgyia pseudotsugata nucleopolyhedrovirus

P35 anti-apoptotic protein from baculovirus

PARP poly(ADP) ribose polymerase

PBS phosphate buffered saline

PCD programmed cell death

PR-3 proteinase-3

PT permeability transition (mitochondrial)

RPV rabbitpoxvirus

RCL reactive center loop

SAPK stress activated protein kinase

SERP2 serine proteinase inhibitor-2 from myxomavirus

SDS sodium dodecyl sulfate

SPI-1 serine proteinase inhibitor-1 from poxviruses

SPI-2 serine proteinase inhibitor-2 from poxviruses

SPI-3 serine proteinase inhibitor-3 from poxviruses

SPI-7 serine proteinase inhibitor-7 from swinepoxvirus

STO mouse fibroblast cell line

TACE TNF-a converting enzyme

TDS transient dominant selection

TIR Toll/IL- IR domain

TLR Toll like receptor

TNF tumor necrosis factor

TRAF TNF receptor associated kinase









variola virus

vaccinia virus

tumor necrosis factor receptor

TNFR associated death domain


VAR

VV

TNFR

TRADD















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLE OF CrmA IN REGULATING INFLAMMATION AND APOPTOSIS DURING
COWPOXVIRUS INFECTION
By

Rajkumar Nathaniel

May 2003

Chair: Richard W. Moyer
Department: Molecular Genetics and Microbiology


Poxviruses are large DNA viruses that replicate exclusively in the cytoplasm of

the infected host cell. The importance of the host inflammatory response in controlling a

poxvirus infection is supported by the fact that approximately one third of the poxvirus

genome is directed at deflecting the early host response to virus infection. Viral serine

protease inhibitors (serpins) are proteins encoded by poxviruses that interfere with the

host immune response. Cowpox virus (CPV) encodes the serpin CrmA which has been

shown to inhibit inflammation and apoptosis during virus infection. Wild type cowpox

virus forms red, hemorrhagic, non-inflammatory pocks on the chorioallantoic membranes

(CAMs) of embryonated chicken eggs whereas CPV deleted for CrmA forms white,

inflammatory pocks accompanied by lowered yield of virus. In vitro studies have shown

CrmA to be a potent inhibitor ofcaspase-1, as well as caspases-8 and -10. Caspase-1 is

an enzyme responsible for the activation of the pro-inflammatory cytokines IL- 1 P and IL-

18 from their inactive precursors. A CrmA-mediated block in the activation of these pro-









inflammatory cytokines would be consistent with the role of CrmA in controlling

inflammation. CrmA also prevents apoptosis, consistent with the demonstrated inhibition

ofcaspases-8 and -10. The myxoma virus serpin, SERP2, has been shown to inhibit

caspases-1, -8 and -10 as does the non-serpin caspase inhibitor, P35, from baculovirus.

However, CPV recombinants where CrmA is replaced with SERP2, P35 or CrmA D303A

(mutation of the reactive center loop (RCL) which alters substrate specificity) all produce

white, inflammatory pocks on CAMs. Virus yields are restored from the P35 and SERP2

but not CrmA D303A recombinants. Pro-apoptotic caspase-3-like activity is detected

only from CPVAcrmA::lacZ and CPVCrmA D303A infections suggesting that loss of

functional CrmA leads to apoptosis and it is apoptosis, not inflammation which is

responsible for the reduction in virus yield. We did not detect caspase-1 within infected

CAMs through processing of chicken proIL-P or proIL-18, suggesting that caspase-1

mediated control of inflammation is unlikely. We show for the first time that the

regulation of apoptosis and inflammation are two distinct functions of CrmA that may not

depend solely on caspase inhibition.














CHAPTER 1
LITERATURE REVIEW

Introduction

Poxviruses are unique DNA viruses in that they replicate exclusively in the

cytoplasm of infected cells. Smallpox, caused by variola virus, is by far the most

infamous poxvirus and has been associated with human infections for about 2000 years

(102). The ability of poxviruses to evade the effectors of antiviral immunity is evidenced

by numerous poxvirus proteins that interact with the host immune system (271).

Studying the interactions of viral proteins would be useful in understanding the immune

response to a poxvirus infection and would also help in elucidating mechanisms of

immune evasion used by poxviruses. Before discussing the role of poxvirus proteins in

modulating the host immune response, it is necessary to review basic aspects of innate

immunity, mediators of inflammation, apoptosis and poxvirus biology.



Immune Systems

The mammalian immune system is divided into the innate and adaptive

components. Innate immunity provides defense against a wide variety of pathogens

without requiring prior exposure, involves an inflammatory response and takes place at

the local site of immune challenge (reviewed in 172, 293). Although innate immunity

was thought to be nonspecific, considerable advances in our understanding of the innate

immune system have made it clear that this response can also recognize self from non-









self. Innate immunity involves a pre-programmed system that includes receptors and the

signaling mechanisms that induce a full inflammatory response that results in up

regulation of proinflammatory cytokines such as tumor necrosis factor (TNF-ca),

interleukin-1 3 (IL-1 P), IL-6 and interferon y (IFNy). As a consequence of

proinflammatory mediator induction, cells such as macrophages, natural killer cells and

neutrophils are activated and migrate to the site of injury. In addition, increased

generation of reactive oxygen species and degranulation of cytoplasmic granules

containing lytic enzymes occur, that aid in the toxic effects associated with these cells.

The adaptive response, on the other hand, requires prior exposure and takes place

in the draining lymph nodes. In the lymph nodes professional antigen presenting cells

(APC) such as macrophages, B-cells and dendritic cells that arrive from sites of

inflammation activate naYve T and B-lymphocytes which then migrate back to the site of

inflammation. Antibodies are produced by activated B-lymphocytes and function to

neutralize the pathogen itself or offer protection against toxins made by pathogens.

Additionally antibodies bound to antigen enable phagocytic cells to ingest and destroy the

antibody coated or opsonized foreign particle. Antibodies bound to antigen can also

activate plasma proteins called complement, which through a cascade of events can lyse

and destroy certain bacteria and viruses. Activating complement also aids in

phagocytosis of pathogens. While extracellular pathogens can be taken care of by the

actions of antibody and complement, the intracellular pathogens are largely destroyed by

functions of the T-lymphocyte cell mediated response.

Interactions between T-lymphocytes and cells presenting antigenic markers drive

the cell-mediated response. Cytotoxic T cells (CTL) recognize virally infected cells and









cause the infected cell to undergo apoptosis or cell death thus preventing viral replication

and release of progeny. Intracellular pathogens taken up by macrophages can evade lysis

by residing in vesicles that have not fused with the lysosome. A subset to T cells termed

T-Helper 1 (TH 1) cells; promote activation of macrophages which allows fusion of their

lysosomes with vesicles thus destroying the invading pathogen. Protection against

extracellular pathogens mediated by antibodies also involves the function of a third

subset of T-lymphocytes termed T-helper 2 cells (TH2). TH2 cells stimulate B-cells to

proliferate and produce antibody to particular antigens. In most cases the adaptive

response confers life long immunity against re-infection by the same pathogen.



Innate Immunity and Inflammation

Inflammation is the reaction of living tissue to any form of injury. Traditionally

inflammation is defined by the Latin words calor, dolor, rubor and tumor meaning heat,

pain, redness and swelling. These physical observations of inflammation are the effects

of cytokines on local blood flow, which allows the accumulation of fluid and cells in the

inflamed area. Primary mediators of inflammation such as interleukin-1 (IL-1) and tumor

necrosis factor (TNF), which are released from injured tissue, cause dilation of blood

vessels around the area of injury. Endothelial cells lining the blood vessels swell and

partially retract allowing the leakage of fluid into the injured area. The increase in body

temperature is a host response to proinflammatory mediators that decreases viral/bacterial

replication and increases antigen presentation by APCs. Migration and extravasation of

leukocytes depend on interactions with the endothelium. Expression of adhesion

molecules such as selections and integrins and their interaction with corresponding ligands









on both leukocytes and endothelial cells control the emigration of leukocytes through

blood vessels. In addition secondary inflammatory mediators such as chemotactic

factors, chemokine IL-8 for neutrophils, are induced during inflammation and serve to

attract leukocytes to areas of tissue injury.

The purpose of an inflammatory reaction is to clear infection, remove dead tissue

and allow access of the immune system to the damaged area. The main cells initially

seen at sites of inflammation are neutrophils followed by the appearance of macrophages.

Activated neutrophils and macrophages undergo the process of respiratory burst whereby

products such as hydrogen peroxide (H202), superoxide radical (02") and nitric oxide

(NO) are produced. The produced reactive oxygen and nitrogen species are released into

the extracellular milieu and act as microbicidal agents. The release of oxygen radicals

and nitric oxide, as well as proteases present in neutrophilic granules such as lysozymes

and acid hydrolases, also leads to localized tissue destruction. In addition, the activation

of clotting pathways as a result of protease action from inflammatory exudates helps in

sequestering the invading pathogen from spread. Lipid mediators such as leukotrienes

(LTB4), prostaglandins and platelet activating factor (PAF) that are released by

phagocytic cells amplify the inflammatory response. PAF is chemotactic to leukocytes

and activates PMNs. Typically the injured area is replaced by tissue identical to that

which was lost. If restitution of the original tissue is not possible due to the severity of

injury, the area is replaced by fibrous, non-specific scar tissue. An acute inflammatory

process can become chronic if the injured area is continuously damaged while being

repaired.






5

Antigen present in the draining lymph nodes activates the adaptive arm of

immunity, and thus during the later stages of an inflammatory response lymphocytes are

involved. There is a delay of 4 to 7 days from the time of injury before the effects of an

adaptive response are seen. This period is sufficient for pathogens such as poxviruses to

replicate and cause systemic spread. Therefore, in such cases the innate immune

response is crucial in controlling infections during the period before an adaptive response

is seen.



Innate Immune Recognition

The mammalian innate immune response has two steps in recognizing invading

pathogens. The first involves recognition of pathogen associated molecular patterns

(PAMPs), and the second step is based on recognition of molecular markers specific for

self (172). PAMPs are conserved products of microbial metabolism that are distributed

and common among a number of pathogens. One example of a PAMP is

lipopolysaccharide (LPS) common to all gram-negative bacteria. The molecules that

recognize PAMPs are termed pattern recognition receptors (PRRs). PRRs include a

number of molecules that can be cytosolic, membrane bound and secreted in eukaryotic

cells. PRRs function to opsonize antigens, activate complement, initiate proinflammatory

and apoptotic cascades. A common PAMP associated with virus replication is the

formation of double stranded RNA that can bind and thereby activate protein kinase R

(PKR) (69). PKR phosphorylates eukaryotic initiation factor eIF-2a which serves to

block viral and cellular protein synthesis, inducing interferon responses and causes

apoptosis of infected cells (450). Double stranded RNA also activates 2'-5'-









oligoadenylate synthase (OAS) which synthesizes 2'-5'-oligoadenylate. The

oligonucleotide activates RNAaseL that cleaves viral and cellular RNA (210) thus

blocking viral replication and inducing apoptosis. Other intracellular PAMPs include

Nod 1, a cellular protein that binds to and regulates procaspase-9, an enzyme involved in

apoptotic induction (167). Nod2, a protein expressed in monocytes, activates nuclear

factor kappaB (NF-KB), a transcription factor associated with inflammation (294). The

complete range of ligands that interact with Nod proteins is currently unknown.

Extracellular PRRs include mannan-binding lectin (MBL) that binds mannose

residues that are abundant on microbial surfaces (111). Serine proteases MASP 1 and

MASP2 are activated by MBL and cleave C2 and C4 proteins, thereby initiating the

complement cascade. C-reactive protein (CRP) and serum amyloid protein (SAP)

function as opsonins by binding to bacterial phosphorylcholine (361). The macrophage

mannose receptor (MMR) facilitates the phagocytosis of bacterial and fungal pathogens

(111). Macrophage scavenger receptor (MSR) binds LPS and protects against endotoxic

shock (409). Other examples of extracellular PRRs are the Toll/Interleukin-1 family of

receptors.



Toll and Interleukin-1 Receptors

Toll and Toll-Like Receptors (TLR)

The Toll/IL- R family of receptors are conserved from flies to mammals and play

a central role in the induction of cellular innate immune responses. Drosophila Toll

ligand has been identified as Spatzle (221). The Drosophila Toll gene product was

originally found to be required for dorso-ventral polarity (139). Toll protein was later








found to control anti-fungal response in flies (221). The intracellular domain of Toll was

found to be homologous to a similar region in human interleukin-1 receptor (IL-1 R)

(293). Homologs to IL-1R have since been discovered, that now belong to the Toll-like

receptor (TLR) superfamily.

TLRs are transmembrane proteins that contain leucine rich repeat (LRR)

extracellular domains and Toll/IL-iR (TIR) cytoplasmic domains. The mammalian IL-1

system will be discussed in further detail later. Similarities exist between the fly Toll and

mammalian IL-1 system. Engagement of both receptors by the respective ligands results

in the activation of nuclear transcription factors. In addition to Spatzle, there also exist

adapter proteins such as Tube and a protein kinase Pelle. To date there are at least nine

Drosophila and ten mammalian TLRs known (172). In addition to the Toll ligand,

Spatzle, there are six other Spatzle-like proteins (281). TLR4, the first mammalian TLR

identified was shown to activate NF-KB and induce IL-1, IL-6 and IL-8 genes (254).

Studies with the mouse homolog of TLR4 demonstrated its role in the recognition of LPS

and gram-negative bacteria (172). TLR2 and TLR5 also recognize bacterial components

such as peptidoglycan, LPS, lipids from trypanosomes and flagellin (150, 362), (53),

(141). TLR2 binding has been shown to cause internalization of the receptor bound

ligand to the macrophage phagosome (141,426). Whether this is an exception or a rule

for most TLRs is to be seen. Experimental evidence also suggests that TLR1, 2 and 6

may function in a cooperative manner (138, 301). This suggests that heterodimerization

of receptors could in theory increase the repertoire of ligand specificities. TLR9 is

thought to reside intracellularly on lysosomes where it recognizes unmethylated CpG

motifs in bacterial DNA and induces THI response (145).









The initiation of signaling by TLRs could occur directly following receptor

engagement as in the case of TLR4 (254) (Figure 1). Alternatively the ligand may need

to be processed prior to receptor interaction as seen with Drosophila Spatzle, which

requires cleavage by serine protease Easter in order to activate Toll (221). The signaling

events following TLR engagement seem to be conserved from flies to mammals.

Signaling by TLR is depicted in Figure 1. Similar to IL-1 binding IL-1R, MyD88, an

adapter molecule that contains a TIR domain, is recruited to the receptor complex where

it causes autophosphorylation of serine threonine kinase IRAK (255). Complex protein

kinase interactions result in the activation and translocation of NF-KB to the nucleus

where it induces expression of inflammatory cytokine genes. TLR2, 5, 6 and 9 activate

NF-KB, while TLR2, 4, 6 and 9 can induce the transcription factor activator protein-1

(AP-1) activity (433). TLR2 has been shown to mediate NF-KB activity via PI3 kinase,

suggesting the involvement of GTP binding proteins (18). TLR2 involvement in

apoptosis has also been demonstrated (13, 14). The fact that mutant caspase-8 could

inhibit TLR2 activity suggests the possibility of direct activation of caspases by TLR2.

The inflammatory cytokines TNF-a, IL- 1 3, IL-18 and IL-8 that are upregulated by TLRs

(254) will be discussed in detail later. The identification of more signaling events

associated with TLRs will bring to light their different roles in the immune response. The

TLR system is similar to mammalian IL-1R system (293).



Interleukin-1 (IL-1)

IL-1 is one of the earliest cytokines produced in response to cellular damage or

infection. IL-1 P is the prototypical proinflammatory cytokine that belongs to the IL-1









family, which includes in addition IL- la and IL-1 receptor antagonist (IL-IRa) (98).

Both IL- I a and IL- I3 are synthesized as 31 kDa precursors that are processed to 17 kDa

mature cytokines by cysteine proteases calpain and caspase-1 respectively (55, 58, 189,

238, 413). ProlL-laot is processed between residues 112 tol 13 by calpain whereas prolL-

1lP is cleaved by caspase-1 first at Asp27 followed by cleavage at Asp 16 to yield mature

cytokine (198, 395). ProlL-laot also contains a nuclear localization signal at residues 79-

86 (448). Interestingly while prolL- 13 has an absolute requirement for processing to be

functional, prolL-la is biologically active as a precursor (58, 253,270). Neither IL-la

nor -P3 contains signal sequence regions that would target these proteins to the secretary

pathway (99). The predominant soluble form seems to be IL-1 3 P, whereas IL-la is

thought to be cell associated (21). IL-Ra is a soluble protein that is able to bind IL-1

receptors but is unable to transmit a signal and is thought to control IL-1 mediated

inflammation (86).

There are two receptors identified for IL-1. The 80 kDa IL-1 receptor type I (IL-

I RI) is responsible for signal transduction (Figure 1), whereas the 60 kDa type II receptor

(IL-1RII) does not deliver any signal and therefore functions to regulate IL-1 activity by

acting as a sink (370). The IL-1RI shares 45% homology with the cytoplasmic region of

Drosophila Toll protein (121). In addition to the TIR domain, IL-1RI and II contain

three extracellular immunoglobulin domains. Initial binding of IL-1 3 to the receptor is

with low affinity. Crystal structures of IL-1RI have revealed that binding of IL-1 p to the

receptor induces a conformational change in the receptor that allows the third Ig domain

on the receptor to wrap around the ligand (357, 435). This conformational change in the








receptor presumably facilitates the docking of IL- R accessory protein (IL- RAcP)

resulting in high affinity binding of IL-1 and the transduction of a signal.

The key signaling molecules activated are NF-KB, AP-1 and members of the

mitogen activated protein kinase (MAPK) family (166, 276,372, 386). The initial

signaling step post receptor engagement involves the association of adapter molecule

MyD88, a protein containing both a TIR domain and a death domain (DD) (50). The

MyD88 TIR is thought to trimerize with TIR domains of IL-IRI and IL-1RAcP in the

signaling complex, similarly to trimerization seen with the TNF-receptor (293). MyD88

recruits IL-1R associated kinases (IRAKs) -1 and -2 (282, 447). Like MyD88, IRAKs

also contain a DD, and it is conceivable that the recruitment of IRAKs to the signaling

complex is via the DDs. Although IRAK is phosphorylated on activation, a catalytically

inactive molecule is still active in signal transduction, suggesting that kinase activity is

not an absolute requirement (197, 245). IRAK functions to recruit TNF receptor

associated factor (TRAF-6), which in turn is responsible for the activation of NF-KB

inducing kinase (NIK) via kinases TAB-1 and TAK-1 (291). Consequently NIK

activates a complex called the signalsome containing IKB kinases (IKK) (42). TRAF-6

can also activate the signalsome via kinases ECSIT and MEKK1 (202, 220). This

complex activation of a number of protein kinases culminates in the activation and

translocation to the nucleus of transcription factors NF-KB and AP-1 (276, 372).

Interestingly NF-KB activation has been associated with both cell death and cell

proliferation (51). In general the state of the target cell determines the cellular pathway

involved. Post receptor signaling involves the activation of kinases of the MAPK family

that results in the activation and translocation of transcription factors. Unlike IL- I3, IL-

















f? .is]MgJ ^MyDW da D








asoiain ofl TRADD, whc recruts RI n TA2 to 2 th[rIetrAK-2lex

Aciaino KKJC ocur thrug th atvatio ofIK inss rbyRPeiae
oli rizo oIKK c s.eeRADD r
W(AK-2


TAI



NF-%:B I -KB-



Figure 1. Signaling by TNF, IL-1R and TLR. (A) TNF binds to TNFRI and causes the
association of TRADD, which recruits RIP and TRAF2 to the receptor complex.
Activation of IKK occurs through the activation of IKK kinases or by RIP mediated
oligomerization of IKK complexes. TNFRI induces cell death when TRADD recruits
FADD and activates caspase-8. (B) IL-1 and TLR receptor complexes which include
MyD88 and IRAKs induce the ubiquitin dependent association of TRAF6. As a result
IKK kinase Taki is activated which phosphorylates and activates the IKK complex.
Abbreviations: TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, TNF receptor
associated death domain; FADD, Fas ligand associated death domain; IKK, I kappa B
kinase; NF-KB, nuclear factor kappa B; IL, interleukin; LPS, lipopolysaccharide; IKK, I
kappa B kinase; IRAK, IL-1 receptor associated kinase, Mal, MyD88 adapter-like,
TRAF, TNF receptor associated factor. From reference 42.








IRa does not bind the third Ig domain on the receptor and cannot induce conformational

change in the receptor that would allow IL-1RAcP docking (357). IL-1 mediated signal

transduction was absent in both IL- 1RI-/ and IL-1RAcP-/- mice, suggesting that both the

receptor and accessory proteins were essential components for signaling (77, 126, 212).

The IL-1RII also has three Ig domains but lacks the TIR domain (252). IL-1RII binds all

three forms of IL-1 (252). The lack of the transducing cytoplasmic TIR domain indicates

that it functions as a negative regulator of IL- 1 activity. Consistent with this role, IL-

IRII has very low affinity for IL-1iRa (398). IL-1RII is expressed highly on lymphoid

and myeloid cells where it can be shed and act as a soluble decoy receptor (73). It was

presumed that proteolytic cleavage of the extracellular domains of membrane receptors

results in the soluble forms of these receptors (83). Indeed TNF-a converting enzyme

(TACE) has been shown to shed IL-1RII from cell membranes (334). Soluble forms of

the type I receptor have also been found in circulation. Patients with rheumatoid arthritis

and sepsis have elevated levels of soluble IL- IRs indicating that these receptors function

to regulate IL-1 activity in vivo (19, 125). In areas of local inflammation, tissue necrosis

can release precursor IL- 1p into the extracellular environment where it potentially can be

processed by proteases other than caspase-1. The maturation of IL-1p3 mediated by

enzymes other than caspase-1 is a consideration that may be important in the results

presented in this dissertation. Both IL-1 RI and II can bind precursor IL-1p3 and prevent

its processing, indicating the importance of soluble receptors in inflammatory fluids

(398).

The fact that biologically active IL- 3P was seen in caspase- 1'/ mice suggested the

existence of other proteases that could process prolL- I P3 (100). This observation may be








important in interpreting the results of experiments presented in this thesis. ProlL-1 3 can

be released from cells by an unknown mechanism despite the absence of any secretary

signal sequences in the molecule (33). In addition, cell lysis can also cause the release of

cytoplasmic proIL-1 p during tissue damage (142). A number ofproteases can be found

at sites of inflammation such as in synovial fluid, sites of neutrophil and macrophage

infiltration and at sites of tissue damage. Chymotrypsin and Stapylococcus aureus

protease are able to process the 31 kDa proIL- 1 3 to biologically active fragments ranging

from 17 to 19 kDa whereas the Trypsin cleavage product is inactive (38). Cathepsin G,

collagenase and elastase can also process proIL-1 P to bioactive cytokine (143). In

addition, the importance of such mechanisms in vivo was evidenced by similar processing

capabilities seen in synovial and bronchoalveolar lavage fluids from patients with

inflammatory polyarthritis and sarcoidosis (143). The CTL molecule granzyme A, a

serine protease, can also generate biologically active IL- I3, although this cleavage occurs

at Argl20, four residues downstream of the Asp 116 site used by caspase-1 (169). The

serine protease chymase, derived from dermal mast cells, also converts proIL-1 p to an

active species, suggesting a role for the initiation of inflammation in the skin (261).

Matrix metalloproteinases (MMPs) have also been implicated in the processing ofproIL-

1 P3 (355). Stromelysin (MMP-3) and gelatinase-A (MMP-2 and -B (MMP-9) can

generate biologically active forms of IL- IP. Extended incubation of the mature product

with these enzymes caused degradation. In addition, none of these MMPs were able to

process proIL- la. Proteinase-3, a proteolytic enzyme from activated neutrophils, has

also been shown to produce IL-1i P (71). The balance of accessible proteases at sites of

local inflammation may regulate the availability of active IL- 1 3 and thus modulate acute








and chronic states of inflammation. In summary, a number of proteases other than

caspase-1 can cleave pro-IL- P13 to generate the active cytokine.

A number of knockout mice models in the IL-1 system has been studied and are

listed in Tables 1 & 2. IL-I a is the least well studied, and observations indicate that it is

not involved in the inflammatory responses induced by turpentine or infection with

Listeria (98, 155). IL-1 P13 is crucial for the development of an acute phase response and

in the induction of febrile response to inflammation (472). Quite unexpectedly IL- 1 P1'

mice were hypersensitive to both IL-a and IL- I3, an observation that so far has not been

explained (12). Mice deleted for IL-Ra were seen to spontaneously develop autoimmune

disorders that resembled rheumatoid arthritis, arterial inflammation and were shown to be

highly susceptible to LPS induced lethality (149, 156, 290). IL-1RI knockout mice failed

to induce a biological response to either IL-1 a or 13, indicating that this receptor is

essential for all IL-1 activity (126, 212). In IL-IRAcP"'/ fibroblasts, binding of IL-l a or

IL-IRa was only moderately reduced, whereas IL- 13 binding affinity was reduced 70-

fold indicating the requirement of accessory protein for efficient receptor binding (77).

Similarly these mice failed to induce a biological response to IL-1. Disruption of the

signaling component IRAK caused reduced activation of NF-KB in fibroblasts and

attenuated responses to IL-1 in mice (410). Caspase- 1' /" mice are deficient in processing

IL-13 (209). Other studies have shown mature IL-1 13 to be released despite caspase-1

deficiency (100). Although caspase-1 I is the most important protease required for IL- 13

maturation other enzymes have been implicated to perform this function. The importance

of enzymes other than caspase- I in IL- 113 maturation may be important in certain cases

and will be discussed in relation to the results presented in this dissertation. These








00I





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(0





I roz




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& .0
'a 9 9 9





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iiIN
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16



Table 2. List of mice deficient in the IL-1 system.


IL-1 system component Mouse strain (reference)
IL-IRI C57BL/6 x 129J (126); C57BL/6 x 129/SV (212)
IL-1RAcP C57BL/6 (77)
IL-la C57BL/6 x DBA/2 (155)
IL-IP C57BL/6 x DBA/2 (155); C57BL/6 x 129EV (100)
IL-lreceptor antagonist C57BL/6 (149,290); MF-1 (290)
IRAK C57BL/6 (410)
Caspase-1 C57BL/6 x 129 (100)









studies underscore the importance of IL- 1 as a key proinflammatory cytokine that

mediates systemic effects and rapidly activates the innate immune response.

The end result of IL-1 signaling is the expression of genes, most of which are

involved in immunity and inflammation (81). IL-1 induces expression of a number of

cell proliferation factors such as granulocyte colony stimulating factor (G-CSF),

granulocyte-monocyte colony stimulating factor (GM-CSF) and monocyte colony

stimulating factor (M-CSF). In addition IL-1 also stimulates platelet production and

increases prostaglandin E synthesis. The production of a number of cytokines involved

in generating an inflammatory response is also stimulated by IL-1 including IL-1, -2, -3, -

6, -7, -8 and TNF. IL-1 can also activate natural killer (NK) cells, T- and B-cells. IL-1 is

a potent fever-inducing agent (81). The activities of IL- 1 demonstrate its role in

inducing an inflammatory response. The crucial role of IL-1 in forming part of the innate

immune response is borne out by the fact that pathogens such as poxviruses encode

molecules that interfere with IL-1 P function. The CrmA protein encoded by

cowpoxvirus is a potent inhibitor of caspase- I thus affecting the IL- I3 maturation

process (118). In addition cowpoxvirus also encodes a (decoy) receptor that binds IL- 13

preventing interaction with cellular IL-1R (381). Both CrmA and the virus-encoded IL-

I OR molecules are the subjects of this dissertation.



Inflammatory Mediators

Tumor Necrosis Factor (TNF)

Along with IL-1, TNF is considered a primary proinflammatory cytokine.

Although mainly produced by mononuclear cells, TNF can also be produced by a variety









of cells including monocytes, macrophages, mast cells, epithelial cells, neutrophils, as

well as T- and B- lymphocytes (277). It exists in two forms, TNF-a and TNF-P3. TNF-a

is synthesized as a membrane-bound 26 kDa protein that is proteolytically cleaved to a

soluble 17 kDa fragment at Val77 by TNF-a converting enzyme (TACE) (39, 273).

TACE is a membrane-anchored enzyme that belongs to the disintegrin-metalloprotease

family and contains an extracellular zinc-dependent protease domain. TNF-P is a 25 kDa

glycoprotein produced by lymphocytes and is also called lymphotoxin-a (130). TNF-a

and TNF-P share 50% homology at the amino acid level (443). Both of the molecules are

active only as homotrimers (24). Although TNF-a and P have similar affinities to the

TNF receptors, they differ in their ability to cause mitogenic and cytotoxic effects (319,

360).

TNF-P is more mitogenic, as its trimers are seen to be stable for several days and

were effective in inducing fibroblast growth that requires extended presence of the

cytokine (358,360). TNF-a trimers are less stable but have a 200-fold increase in

cytotoxicity over TNF-P3 (360). Gene disruptions in both TNF-ac and -P3 protected mice

from zymosan-induced organ dysfunction, indicating the involvement of these cytokines

in inflammation (436). Both TNF-ca and 3 are able to interact with two receptors that

mediate their biological effects (152). Binding to the receptors results in a wide range of

biological effects including cell proliferation, tumor necrosis, inflammation and

apoptosis, which are dependent on, cell condition and type of associated molecules

recruited to the signaling complex. TNF-ca is very effective in controlling local infection.

However, systemic release of TNF-a is detrimental to the host leading to septic shock








and ultimately death by multiple organ failure due to increased vascular permeability

resulting in loss of blood volume and plasma clotting proteins.

The TNF receptor, TNF-RI, has a molecular weight of 55 kDa and acts as the

main receptor type expressed by most cells (24). TNF-RI is thought to mediate most of

the proinflammatory effects of TNF-a (315). TNF receptor mediated signaling is

depicted in Figure 1. The intracellular domain of TNF-RI contains a death domain (DD)

that is required for apoptotic signaling of cell death (402). TNF-RI is always associated

with silencer of death domains (SODD), a cellular protein that functions to prevent the

inadvertent self-association of TNF-RI death domains (173). Following TNF binding to

TNF-RI, the receptor trimerizes leading to the disassociation of SODD. The adapter

protein TNF-R associated death domain (TRADD) interacts with TNF-RI via the DDs

(159). The Fas associated death domain (FADD) adapter protein that interacts with Fas

during apoptotic signaling does not directly bind TNF-RI, but can be recruited by

TRADD to the TNF-R signaling complex. (160). The association of FADD leads to the

recruitment of procaspase-8 followed by its activation and the initiation of the caspase

cascade.

The N-terminal domain of TRADD has been found to interact with the C-terminal

domain of TNF associated factor (TRAF) 2 (160). TRAF-2 plays a role in the activation

of NF-KB and Jun N-terminal kinase (JNK) by both receptors but is not involved in

apoptosis (161,464). RIP, a cytoplasmic serine/threonine protein kinase, strongly

interacts with TRADD in the receptor-signaling complex and activates NF-KcB and JNK

pathways (159, 232). However RIP disruption (RIP/-) affects only NF-KcB but not JNK








activation (190). Thus is seems that TNF-RI signaling of NF-KB is mediated via RIP

while TRAF-2 enables JNK activation.

TNF-RII is a 75 kDa receptor that is co-expressed with TNF-RI predominantly in

endothelial and blood cells and is involved in passing bound TNF to TNF-RI for

signaling (151, 403). In addition, TNF-RII can directly signal NF-KB activation (152).

TNF-RII has been found to associate with a number of proteins including TRAF-1,

TRAF-2, cellular inhibitor ofapoptosis protein (clAP) -1 and cIAP-2 (343). While

TRAF-1 and -2 can form heterodimers, only TRAF-2 contacts the receptor directly (345).

Similarly clAP-1 and -2 interact indirectly with the receptor by associating with TRAF-1 I

and -2 via their N-terminal BIR (baculovirus IAP repeat) domain (343). Cellular IAPs

have been shown to inhibit the induction of apoptosis by interacting with caspases-3 and -

7 directly as well as target these proteases to degradation by ubiquitination (163, 346).

Cellular lAPs have also been reported to direct the ubiquitination of TRAF-2 (227). It is

unclear at present if clAPs have any role in the activation of NF-kB directly. Expression

of TRAF-2 is sufficient to induce NF-rB and JNK activation under certain conditions

(232,344).

A small subset of TNF binding proteins are the soluble TNF receptors that

represent the extracellular cytokine binding domains that have been shed from the cell

surface by TACE mediated proteolytic cleavage (76, 379). The soluble receptors function

to neutralize the biological activity of TNF by antagonizing the binding of cellular

receptors (94, 179, 364, 379, 406). The outcome of TNF activity depends on the

composition of the signaling complex and can either lead to the induction of apoptosis or

activation of JNK and NF-kB pathways that function to mediate cell survival (232).









In summary both TNF-a and -P are able to bind TNFR and signal via DD

containing proteins initiating a cascade of events leading to the induction of apoptotic cell

death whereas events leading to IKB phosphorylation results in the translocation of NF-

KB to the nucleus where it can induce transcription of genes involved in cell survival and

the proinflammatory response. In addition TNF-oa also causes neutrophil and

macrophage activation and induction to synthesize nitric oxide (NO). TNF-a also

induces the synthesis of acute phase proteins such as C-reactive protein that opsonize

microbes and activate the complement pathway. Mannan-binding lectin (MBL), a PRR

that opsonizes microbes and activates complement is up regulated by TNF-ot. Like IL-

13, TNF-a is also a fever-inducing agent. The important role of TNF, in the innate

immune system and specifically in controlling viral infections, is evidenced by pathogens

such as poxviruses encoding molecules that interfere with TNF function. Cowpoxvirus

encodes five different TNF receptor family homologs that will be discussed in more

detail later.



lnterleukin-18 (IL-18)

IL- 18 was originally discovered as the factor that induced interferon gamma

(IFNy) thus being critical for the control of intracellular pathogens (296). IL-18 is a

potent inducer of CTL and NK cell activity and promotes the differentiation ofT cells to

the THI phenotype (396). While the induction of IL-18 is not entirely clear, it is seen to

be involved in an immune prophylactic response to a number of bacterial and viral

infections (35, 318). Like prolL-13, prolL-18 does not contain a signal peptide sequence,

is intracellular and is processed by caspase-1 (122, 133). The precursor is synthesized as








a 24 kDa peptide that is cleaved at Asp36 to generate the mature 18 kDa cytokine (7).

There exist alternative processing mechanisms that will be discussed later. Unlike IL-1 P,

prolL-18 is constitutively expressed by a variety of cells including Kupffer cells,

macrophages, osteoblasts, keratinocytes, dendritic cells, astrocytes, microglial cells, T

and B cells (99, 285). A molecule purified from urine was shown to bind IL-18 (IL-

18BP) and has been the focus for potential immunotherapy (6,292). IL-18BP was shown

to inhibit the IL-18 induction of IFN y. Interestingly a number ofpoxviruses also encode

a soluble vIL- 18BP underlining the importance of IL- 18 mediated antiviral response (52).

IL-18BP prevents IL-18 from binding its receptor and thus functions as a soluble decoy

receptor to regulate IL- 18 activity.

The 60 to 100 kDa receptor for human IL-18 was originally identified as the IL-1

receptor related protein (IL- 1 Rrp) and is now termed IL-1 8R/IL-18Ra has low binding

affinity for IL-18 (308, 416). Other studies have shown that T cells exhibit high affinity

binding for IL-18 suggesting the presence of a second molecule involved in the binding

complex (466). Subsequently an accessory protein-like (AcPL) receptor that is required

for high affinity binding of IL-18 and transduction of signal was identified (44).

Following binding of IL-18 to IL-1 8R, AcPL also termed IL-1 8R accessory protein

(AP)/IL- 18RP3 is recruited to the receptor complex where it forms a high affinity

heterodimer (336, 369). IL-18R3 does not bind IL-18 by itself and does so only after IL-

18 is bound by low affinity to IL-18Ra (44). Although proteolytic processing of the

extracellular receptor domains can result in soluble forms of both molecules, only soluble

IL-18Ra (s IL-18Roa) binds IL-18 it does not neutralize the effect of IL-18. The

enhancement of affinity for IL- 18 binding to IL-I 8Ra is supported by recent evidence









showing that soluble IL-1 8Rca only in combination with sIL-18Rp3 is able to block IL-18

induction of IFNy (336). Thus the regulation of IL-18 by soluble forms of the receptors

is similar to that seen in IL-1.

The effects of receptor binding and signal transduction are similar for both IL-18

and IL-1. IL-18 signaling complex (comprising of IL-18 bound to IL-18Ra and IL-

18RP3) recruits IRAK via the adapter protein MyD88 (340, 411). MyD88"/- cells do not

respond to IL-18 (1). Upon activation, IRAK dissociates from the receptor complex and

is responsible for the activation of NIK mediated by TRAF-6 (200, 411). The signalsome

containing IKB kinases (IKK) is activated by NIK directly or alternatively by TRAF-6

activation of MEKK1 and ECSIT (42,202, 220). The complex interaction between a

number of protein kinases culminates in the activation and translocation of transcription

factors NF-KB and AP-1 (25, 248, 340). The steps leading to the activation of NF-KB

and AP- 1 seem to be identical both in IL- I3 and IL-18. IL-18 is also involved in

activating the MAPK pathway. IL-18 induced the kinase activities of MAPK and LCK in

THI cells (419). MAPK is involvement in cell growth could possibly explain the effect

of IL-18 in activating NK and T cells (415). Apart from inducing IFNy production from

T, B and NK cells, IL-18 acts in synergy with IL-12 to induce NK cell cytotoxicity and

THl cell differentiation (35). Signaling pathways used by IL-18 and IL-12 are quite

different. While IL-18 activates NF-KB, IL-12 activates the signal transducer and

activator of transcription (STAT) 4 (25). The lack of IFNy production by IL-18 observed

in TH2 cells may be due to the fact that TH2 cells do not express IL-1 8R (466). In

addition to IFNy, IL-18 is a potent inducer of the chemokines MIP- lac, MCP-1, IL-8, as

well as IL-lP3 and TNF-a production in different cell types (321). The ability of IL-18 to









induce an innate response as well as stimulate the production of a number of

inflammatory cytokines underscores its importance as a proinflammatory mediator.

Both IL- 18 and IL- 18R have been targeted for gene disruptions. IL- 18 deficient

mice have impaired IFNy production, defective NK cell activity and reduced THI

response (399). The importance of IL-18 in controlling intracellular pathogens was

demonstrated in IL-18-/' mice infected with the protozoan parasite Leishmania that were

highly susceptible to infection compared to wild type controls (445). Infections with the

extracellular bacteria Stapylococcus aureus caused reduced septicemia whereas these

mice developed more severe septic arthritis, a reaction associated with impaired THI

response (445). Receptor knockout mice have also been characterized. IL-18"-' cells

from these mice had defects in NK cytolytic activity, IFNy production and were impaired

in NF-KB or JNK activity (157). The IL-1 8R-/ mice also failed to respond to IL-18

indicating the essential role of this molecule in mediating signaling.

The availability of mature IL-18 under diverse conditions can determine the

outcome of the immune response. While IL-18 is processed to maturity by caspase-1 at

Asp36, there also exist alternative processing mechanisms. In addition to caspase-1,

caspases-4 and -5 have also been shown to process prolL-18 correctly (122). Caspase-3

on the other hand processed IL- 18 at Asp71 and Asp76 resulting in polypeptides with no

significant biological activity (7, 133). Proteinase-3 (PR-3), a serine protease present in

neutrophil granules has also been shown to process prolL-18 (99, 389). Both membrane

bound and soluble forms of PR-3 enzyme have been identified. PR-3 has been associated

with a number of inflammatory diseases and has recently been implicated as one the

major autoantigens in Hepatitis C virus infection (47, 99, 348, 453). Interestingly PR-3









can also mediate the induction of apoptosis in HL-60 cells that is independent of caspase

activity and loss of mitochondrial membrane potential in spite of being implicated in the

cleavage and activation of caspase-3 (453). Of interest will be future studies on

understanding the biological activities of PR-3 since it seems to play a role in both

inflammation and apoptosis.

In summary, IL- 18 induces the production of inflammatory cytokines (TNF-oc,

IL-113 and IFNy), growth factors (GM-CSF) and chemokines (IL-8, MCP-1, MIP-la and

MIP-1 3). The biological activities associated with IL-18 drive a THI response, enhance

NK cell cytotoxicity and induce macrophage activation. Thus IL-18 plays a crucial role

in the innate immune response against pathogens such as viruses. The fact that

poxviruses such as cowpoxvirus encode molecules to bind IL-18; thereby disrupting

cytokine function, indicate the importance of IL- 18 as an antiviral agent and mediator of

inflammation.



Interleukin-8 (IL-8)

IL-8 was originally identified as a neutrophil chemo attractant from supernatants

of activated monocytes (359, 437,467). IL-8 is a 7 tolO kDa protein that belongs to the

CXC family of chemotactic cytokines termed chemokines and is now called CXCL8.

Chemokines are classified based on the position of the first two N-terminal cysteines that

can be separated by one amino acid (CXC) or can be adjacent (CC) (22). Disulphide

bonds between the first and third Cys as well as the second and fourth Cys are important

for three dimensional folding, receptor recognition and biological activity. IL-8 is

synthesized as a 99 amino acid precursor that is proteolytically processed by the removal








of a 20 amino acid leader sequence (67). Maturation of IL-8 occurs extracellularly with a

number ofproteases being implicated in the cleavage process. Two forms of mature IL-8

have been observed to be secreted from activated neutrophils IL-8 [77] and IL-8 [72]

containing 77 or 72 residues respectively which can be further processed by neutrophil

elastase, cathepsin G and proteinase-3 to give N-terminal truncated forms that are more

active (302). Neutrophil gelatinase B, a secreted matrix metalloproteinase (MMP-9) has

also been shown to cleave mature IL-8 [77] to more active truncated forms (431). Thus

inflammatory exudates containing proteases can potentiate more active forms of IL-8.

Near sites of production, chemokines can form oligomers on endothelial or

extracellular matrix, thereby forming a gradient with higher concentrations of chemokine

near the inflammatory stimulus (298). In addition to attracting neutrophils, IL-8 has also

been shown to have chemotatic effects on a number of other cells including basophils,

eosinophils, T lymphocytes and macrophages (366). The ability of IL-8 to attract

neutrophils has been disputed by both in vitro and in vivo experiments involving

intradermal injections in humans (218, 222, 405). In addition to being secreted by

neutrophils, IL-8 is also secreted by keratinocytes, fibroblasts, endothelial cells and T

cells (310). IL-8 has been demonstrated to have mitogenic effects (353). Subsequently

constitutive IL-8 expression has been observed in many types of human cancers (366).

IL-8 has also been shown to induce angiogenesis (199).

Although monomeric IL-8 is biologically active, IL-8 can also exist as a dimmer

with the disulphide bonds playing important roles in anchoring the cores of the dimmer

together (70). N-terminal residues Glu4, Leu5 and Arg6 (ELR) have been shown to be

absolutely essential for receptor binding (67, 144). The ELR motif is found to be present








only in chemokines that attract neutrophils (22). Synthetic IL-8 containing modifications

of the ELR sequence can function as receptor antagonists that block IL-8 action

suggesting that N-terminal truncations that occur naturally during inflamed states could

contribute to regulation (268). Two IL-8 receptors have been identified, the CXCR1 and

CXCR2 (154, 280). The receptor more selective for IL-8 is CXCR1 (269). Chemokines

exert their function via heterotrimeric seven transmembrane G-protein coupled receptors

(GPCRs) (22). The functions of the two receptors are also different in certain aspects.

While both receptors induce cytosolic free Ca2 and degranulation, CXCR1 mediates

respiratory burst and the activation of phospholipase D (181).

Initial studies on chemokine signaling were performed in human neutrophils

stimulated with IL-8 (22, 298, 310). The functional responses were found to be similar

for all chemokine receptors. Following chemokine binding the receptor, there is a

conformational change in the chemokine at the N-terminus with disulphide bonding

between Cys7 and Cys34 (1st and 3rd Cys bond) playing a major role in receptor binding

and activation (325). The conformational change facilitates further interaction with the

receptor that leads to activation. Activation leads to the exchange of bound GDP for GTP

in the G-protein. GTP bound ca subunit and P3y dissociate. The signal transduction

enzymes phospholipase C (PLC) and phosphatidylinositol-3-OH kinase (PI3K) are

activated by P3y subunit. PLC mediates cleavage of phosphatidylinositol (4,5)-

bisphosphate yielding inositol triphosphate (IP3) and diacylglycerol (DAG). IP3

induction results in release of intracellular stores of calcium giving rise to a transient

increase in free calcium concentration. DAG activates protein kinase C (PKC). PI3K

also generates phosphatidylinositol triphosphate and activates protein kinase B (PKB).









The Py subunit also brings about activation of MAP kinase and then re-associates with

GDP bound a subunits. This complex activation of kinase pathways results in the

activation of transcription factors that up regulate the expression of cell adhesion

molecules, growth factors and inflammatory cytokines that facilitate the motogenic and

mitogenic effects of IL-8.

In neutrophils, the transient increase in cytosolic free calcium and activation of

PKC is responsible for granule release and superoxide production (23). The activation of

GTP'ases results in cytoskeletal restructuring (40, 219). CXCR1 mediated activation of

phospholypase D also leads to superoxide formation in human neutrophils (181, 211).

Activation of map kinases leads to the up regulation of transcription factors (180, 414).

Binding of ligand to CXCR2 can also lead to receptor desensitization mediated by PKC

followed by receptor endocytosis and degradation, a mechanism thought to regulate IL-8

(278,279, 461). While the activation of transcription factors AP-1 and NF-KCB by TNF-a

and IL-I 3 up regulate IL-8, expression of IL-8 is inhibited by interferons a and 13 (262,

371). Since IL-1 3 and TNF-a are elevated during inflammatory states and the fact that

IL-8 is synthesized by a number of cell types, highlights its important in causing local

accumulation of neutrophils in any tissue.

In summary, IL-8 functions as a chemotactic factor for leukocytes increasing

access of immune cells such as neutrophils to sites of inflammation. IL-8 enhances

adhesion molecule binding (132 integrin) to circulating leukocytes thereby facilitating

migration and extravasation of the immune cells across blood vessel walls into inflamed

tissue. IL-8 activates neutrophils to undergo respiratory burst and degrannulation. In








short, IL-8 is particularly important in the innate immune response by directing

neutrophil migration to sites of infection.



Avian Cytokines

A number of homologs for mammalian proinflammatory cytokines have been

identified in birds. A review of avian and mammalian cytokines reveals that even though

they function similarly, some of their primary structures seem to differ significantly.

Avian homologs of TNF-a, IL- 3P, IL-18 and a number of CXC chemokines have been

identified so far. A majority of these molecules were discovered in chickens, in addition

to duck, turkey, pheasant, quail and guinea fowl (384).



TNF-a

TNF-a-like activity can be detected in chick embryos as early as day 1 (452).

The pattern of expression throughout the growing embryo suggested a role for TNF in

apoptosis during differentiation of the neuronal system as well as in tissue remodeling.

Indeed cells undergoing DNA fragmentation associated with apoptosis were

demonstrated to be closely associated with TNF-a immunoreactivity (451). Chicken

macrophages stimulated with bacterial LPS or the intracellular protozoan Eimeria,

secreted a TNF-like activity that was preferentially cytotoxic to chicken fibroblasts

indicating its role in bacterial and coccidial infections (471). Partial purification of

stimulated macrophage supernatant fractions containing TNF-like activity revealed a 17

kDa protein that cross reacted with antibodies to human TNF-a (329). Although the

partially purified protein induced biological activities related to TNF-ca in chicken









macrophages, it was not blocked by antibodies to human TNF-a suggesting structural

differences and/or the presence of other factors in the preparation. Chicken TNF-RI has

recently been cloned and was found to be 47% identical to its human counterpart in the

cytoplasmic death domain (46). In addition a number of death receptors (DRs) related to

TNF-RI have been identified in chickens that are yet to be fully characterized (46).



IL-1

Chicken IL-1 activity was first seen in LPS stimulated spleenocyte supematants

(140). Chicken macrophages treated with LPS also induced secretion of IL-1 activity

(41). The induction of IL-1 was seen to be dependent on protein kinase C and a

calmodulin dependent kinase. Other studies have also shown that IL-1 secretion by

macrophages is dependent on intracellular calcium levels (65). IL-1 also lowered growth

rates in chicken similar to the effects seen with inflammatory agent Sephadex (195). The

chicken IL- IP gene was cloned rather recently and a peptide containing residues 106 to

267, the putative mature IL-P sequence, was shown to be biologically active both in vitro

and in vivo (446).

Unlike the mammalian system where maturation of IL-1 P requires caspase-1 to

process the inactive precursor initially at Asp27 (site 1) followed by cleavage at Aspl 16

(site 2), the chicken proIL- 113P sequence does not contain either of the conserved Asp

residues required for cleavage that would release the mature cytokine (395, 446). The

mechanism of IL-1 P13 maturation in chickens is currently unknown. It remains a

possibility that in chickens, IL-1 13 does not require processing and maybe active as the

precursor itself. The chicken proIL- 113 sequence is similar to proIL- 113 sequences from








frog and fish, which also do not contain the conserved Asp sites found in mammals (475).

Matrix metalloproteinases (MMPs) have also been implicated in the processing of

mammalian proIL-1P (355). Gelatinase A (MMP-2) and B (MMP-9) are among the

MMPs shown to cleave prolL-13. An avian gelatinase that shares homology with MMP-

2 is observed to be over expressed in chicken embryo fibroblasts transformed with Rous

sarcoma virus (64). In addition, a chicken enzyme similar to MMP-9 has been cloned

from macrophages that differ from its mammalian counterpart in sequence and

biochemical function (137). In light of alternative mechanisms of processing observed

for mammalian IL- 1p, it is worth noting that chicken heterophils treated with LPS,

zymosan or phorbol myristate acetate (PMA) show increased MMP production (328).

Although there seem to avian homologs to mammalian enzymes that have been

implicated in IL- l3 maturation, there is no experimental data to prove that any of the

avian enzymes actually function to process avian IL- 1P3.

The chicken IL-1RI was cloned from chicken embryo fibroblasts (136).

Interestingly the cytoplasmic region of chicken IL-1RI is nearly 80 % homologous to

both its mammalian counterpart as well as the Drosophila Toll receptor. Like other TLRs

the chicken receptor also has an extracellular Ig-like region although sequence

divergence is higher in this region. Soluble chicken IL-1RI was shown to inhibit IL-1-

like activity released from LPS induced macrophages confirming an antagonistic role for

this molecule (196). The chicken homologs of IL- la, IL-1 RII or IL- IReceptor

antagonists are yet to be identified.









IL-18
The chicken IL-18 gene was discovered as a result of BLAST search analysis of a

bursal expression sequence tag (EST) database (354). Comparison of the primary

sequence between the chicken and mammalian homologs revealed that the chicken

precursor contains the conserved Asp36 that is required for processing by caspase-1 (7).

A putative peptide representing residues 30 to 198 expressed with an N-terminal

histadine tag was found to induce IFNy synthesis in primary chicken spleen cells. The

IL-18 receptor and accessory signaling protein as well as the antagonist IL-18 binding

protein are yet to be identified in birds.



CXC chemokines

The chicken chemotactic and angiogenic factor (cCAF) is a 9 kDa CXC

chemokine that was originally cloned from Rous sarcoma virus transformed fibroblasts

(29, 388). Recent studies have suggested it to be a homolog of IL-8 (183). Similar to IL-

8, the chicken cytokine can also be post translationally cleaved (by plasmin) to smaller

fragments (243). Experimental evidence points to the C-terminus as being angiogenic

(243). K60 is another chicken CXC chemokine that shares 50% homology to mammalian

IL-8 (367). K60 is secreted from macrophages stimulated with LPS, IL-1l and IFNy

(367). Interestingly both cCAF and K60 are the only chicken chemokines to date that

contain the conserved ELR motif present in all mammalian chemokines that attract

neutrophils (22). A homolog of mammalian CXCRI was cloned from a chicken genomic

library (226). The chicken receptor was found to be 67% homologous to human CXCRI.

The identification of mammalian homologs of IL-8 and related CXC chemokines in birds

demonstrate the similarities between the chemotatic systems.









Viral Inhibitors of Inflammation

The outcome of viral infections depends on both host and viral factors. The early

resolution of infection is the goal of the mounted innate immune reaction. Not

surprisingly, viruses have been shown to encode a number of molecules that interfere

with the early host innate response. Viral products that specifically function to abrogate

proinflammatory cytokines and their effects will be discussed here. Cytokine synthesis is

a typical early host response to viral infection. Different viruses target the same cytokine

pathways underlining their importance as potent anti-viral agents and these include TNF-

a, IL- 1 P, IL- 18, the interferons, complement as well as a number of chemokines. The

strategies employed by viruses function to disrupt cytokine transcription/up regulation,

interfere with receptor binding and modulate cytokine signaling.



TNF Inhibitors

Encoding decoy receptors that bind TNF is a mechanism employed by a number

of poxviruses and herpesviruses. Shope fibroma virus encodes the T2 protein that has

been shown to be a TNF receptor homolog that binds both TNF-a and P3 (249). A

homolog to the T2 protein was also found in Myxoma virus (427). Deletion of Myxoma

virus T2 had no effect on growth of the virus in tissue culture but significantly attenuated

viral lethality in infected rabbits. Cowpoxvirus encodes five different TNF receptor

family homologs CrmB, CrmC, CrmD, CrmE and a soluble CD30 homologue. The

CrmB protein shares homology to TNFRII in the ligand binding region and can bind both

TNF-a and 3 (162). CrmC also shares homology to TNFRII but only binds TNF-a

(375). CrmD found only in some strains of cowpoxvirus and ectromelia virus was able to









bind both TNF-a and P and block their cytolytic activity in vitro (233). In addition to

being present in cowpoxvirus, CrmE is also present in other poxviruses such as vaccinia

virus (352). The cowpox CrmE was also found to only inhibit TNF-ca activity. More

recent work has shown the vaccinia CrmE protein to also bind TNF-a but not P3 (333). In

this study CrmE was found to inhibit the cytotoxic activities of human TNF-a but not

those of mouse or rat. Vaccinia strain USSR deleted for CrmE was attenuated in an

intranasal mouse model of infection. But vaccinia WR strain, engineered to either

express the USSR CrmE, or CPV CrmB or CPV CrmC or CPV CrmE genes displayed

enhanced virulence indicating the influence of viral TNFRs on disease outcome. The

CD30 molecule is a member of the TNF receptor family and is associated with TH2

responses although its exact function role is unclear at present (31). The important role

of CD30 in viral infection is evidenced by the fact that cowpoxvirus encodes a secreted

CD30 homologue. Another virus that interferes with TNF is the herpes human

cytomegalovirus that encodes UL144, which also belongs to the TNFR family (30).

While the ligand that binds UL144 is yet to be identified, its importance in viral infection

is demonstrated by the presence of antibodies to UL144 in patient sera.



IL-1 Inhibitors

Members of poxviruses also encode molecules that interfere with IL- 113 either in

its production or signaling. The subject of this dissertation work is the cowpoxvirus

CrmA protein that will be discussed in more detail later. Briefly, CrmA is believed to

inhibit the processing of proIL-113 by blocking the activity of the processing enzyme,

caspase-1 (331). The importance of IL-1 3 involvement in viral infection is further









evidenced by the expression of soluble IL- I3 receptors encoded by poxviruses. The

vaccinia virus B1 5R protein based on sequence was proposed to function as an IL-1

binding protein (376). Indeed B15R was found to bind IL-113 but not IL-1-at or IL-IRa

(8,381). Viruses lacking B15R were found to be less virulent than wild type when

injected into weanling mice via the intracranial route. Similar experiments in mice

infected via the intranasal route with virus disrupted for B15R accelerated the illness and

mortality indicating a role for IL-1 in modulating virulence. Furthermore B15R was also

found to prevent fever caused by virus infection in mice. When the non-functional B15R

gene in Copenhagen, a virulent strain of vaccinia, was repaired, the resulting virus

infection suppressed fever and attenuated viral virulence (10). Recent evidence shows

that poxviruses can also interfere with signaling events downstream of TNFR and IL-1 RI

such as NF-KB activation although observed responses seem to be specifically different

for each virus tested (295).



IL-18 Inhibitors

The central role of IL- 18 in inducing IFNy that protects against bacterial and viral

infection is well documented (318). Interestingly a soluble cellular IL-18 binding protein

(IL-1 8BP) was identified and found to be more homologous to putative poxviral proteins

than to mammalian IL-18 receptors (292). In the cellular context IL-18BP functions as a

decoy receptor to bind IL-18 preventing its activity. Viral IL-18BPs (vIL-18BP) have

been identified in vaccinia, cowpox, ectromelia, variola and swinepox viruses (292). In

addition Molluscum contagiosum virus was found to contain three open reading frames

with homology to IL-18BP (457). Two of the Molluscum gene products MC53L and









MC54L bound both human and murine IL-18 with high affinity and prevented IL-18

mediated IFNy induction in KG-1 monocytic cells. IL-18 binding activity was found in

culture supematants of a number of vaccinia strains as well as cowpox and ectromelia

viruses (377). Furthermore vIL-18BP of ectromelia virus was found to inhibit NF-KB

activation and IFNy induction in KG-1 monocytic cells in response to IL-18. Cowpox

and ectromelia viruses are rodent pathogens and not surprisingly their vIL- 18BPs

preferentially bound mouse IL-18 over human IL-18 (52). Mice infected peritonealy

with ectromelia deleted for vIL-18BP had increased NK cell cytotoxic activity while the

mutant virus was found to be attenuated (43). IL-18 activity clearly is an important host

response mechanism seeing that a number of viruses block its activity.



Chemokine Inhibitors

The number of mechanisms employed by different viruses to subvert chemokine

activity indicates the importance of this response to control viral infection. Similar to

counter strategies used against cytokines, different herpes and poxviruses encode

molecules that regulate chemokines. Typically these fall into three categories. First are

molecules that are ligands or viral homologs of cellular chemokines. These include

human herpes virus proteins such as vMIP-I, vMIP-II, vMIP-III, that are homologs to

MIP-I a or P3 and function as agonists or antagonists (213,264, 385). Cytomegalovirus

proteins vCXC-1 and -2 as well as Marek's disease virus vIL-8 are homologs of IL-8

(307, 312). Interestingly vIL-8 was found to chemoattract blood mononuclear cells but

not heterophils. Molluscum encodes two proteins that act as antagonists to MCP-I. The









MC148R molecule is able to bind receptors but does not transmit signal as it lacks a

region critical for receptor activation (78, 206).

The second group of viral proteins is chemokine receptor homologs. Virus

encoded chemokine receptor homologs are typically cell membrane bound and function

to bind chemokines. These include the ORF74 protein found in a number of herpes

viruses that binds CXC chemokines (342). The US28 gene ofcytomegalovirus is a

functional CC chemokine receptor (36). In addition this virus also encodes US27, UL33

and UL78 proteins with homology to CC chemokine receptors some of which have been

found to localize on viral membranes (109, 240, 330). Swinepox virus encodes the K2R

molecule that is potential receptor of CXC chemokines (246) (Traci Ness and Richard

Moyer unpublished). Capripoxvirus also contains a putative CC chemokine receptor the

Q2/3L ORF (54). Molluscum encodes MC148, a MIP-1 P3 homolog that was shown to

antagonize the chemotactic activity of a number of CC and CXC chemokines on different

cell types (78).

The third mechanism employed by viruses is to secrete binding proteins that

sequester chemokines and prevent chemotaxis of inflammatory cells to the site of

infection. Examples of these would include the myxomavirus M-T7 protein that was

originally discovered as an IFNy receptor but has subsequently been found to also bind C,

CC and CXC chemokines (214,429). The M-T1 gene product of myxomavirus and

rabbitpoxvirus secreted 35 kDa proteins function similarly in binding CC chemokines

with high affinity and IL-8 (CXC) with a lower affinity but share limited homology (129,

216). In vivo studies indicate that M-T1 deletions in myxoma do not affect virulence but

the absence of M-T1 increased infiltrating monocytes and macrophages at the site of









primary infection (215). More recently M-T1 was found to differ from rabbitpoxvirus 35

kDa protein in that M-T1 I can localize to cell surfaces through interactions at the C-

terminus of the molecule and heparin (363). The breadth of viral modulators of

chemokines suggests that chemotaxis is an important host response to virus infection.



Inhibitors of Complement

Activation of complement pathways can itself lead to the destruction of virus-

infected cells as well as mediate the release potent inflammatory cytokines and is another

mechanism targeted by virus infections. Mammalian regulators of complement

activation function to protect bystander cells from the effects of activated complement.

These regulators are characterized by homologous motifs termed short consensus repeats

(SCRs) (335). The vaccinia complement control protein (VCP) contains four SCRs and

regulates complement by binding to C4b and C3b (204, 251). VCP acts as a cofactor to

the cellular complement regulator serine protease factor I and aids in the cleavage of C4b

and C3b to inactive components (349). The cowpox VCP homolog called

immunomodulatory protein (IMP) was also shown to be functional in inhibiting tissue

damage due to complement at the site of viral infection both in the footpad and

subcutaneous models of mice injections (158, 258). More recently the variola homolog

of VCP termed smallpox inhibitor of complement systems (SPICE) was found to be

nearly 100 fold and 6 fold more potent at inactivating human C3b and C4b respectively

than its vaccinia counterpart (341). Glycoproteins C (gC) of human herpes virus 1 and 2

have also been found to bind C3 components of complement (203). In addition the

herpesvirus saimiri CCPH gene product prevents complement mediated cell damage and









has been found to bind complement component C3d (108). Clearly inhibiting

complement activation and lysis of infected cells allows viruses more time to replicate

and spread.



Inhibitors of Interferons

In addition to the blocking the mentioned immune mechanisms, viral products

also mediate interferon activity and will be briefly discussed here. Poxviruses encode a

number of proteins to bind both interferon type I and II. A soluble type I IFN receptor

has been identified as the vaccinia B18R open reading frame (72, 397). B18R has high

affinity for IFNca of different species, can also be found membrane bound and its absence

from vaccinia attenuated infections in the murine intranasal model. The myxoma M-T7

products are soluble type II IFN receptors that sequester IFNy (274). A unique feature of

M-T7 is that in addition to binding IFNy it also binds IL-8 and other members of the C,

CC and CXC chemokines (214). Soluble IFNy receptor homologs appear to be present in

ectromelia, cowpox and rabbitpox viruses as well (275). Indeed the vaccinia B8R protein

binds IFNy from a different species (9). Another unique molecule is the 38 kDa

glycopeptide secreted by tanapox virus (95). Unlike any other protein known to date, the

38 kDa protein binds IFNy, IL-2 and IL-5 demonstrating yet another way viral proteins

disarm multiple immune pathways with the same molecule. There also exist numerous

protein molecules encoded by different viruses that regulate the intracellular activities of

IFN that will not be discussed here. Blocking IFN and its activity is vital for virus

survival as evidenced by numerous viral strategies discovered to date.









Apoptosis

Apoptosis is a crucial step necessary for the clearance of infected cells and

activated neutrophils, to protect areas adjacent to the inflammatory tissue from injury.

Apoptosis or programmed cell death is a distinct mechanism by which cells die and are

dismantled in an orderly fashion (191). This process is a distinct morphological and

biochemical function that involves cell shrinkage, membrane blebbing, degradation of

proteins, chromatin condensation, DNA fragmentation followed by disintegration of the

cell into multiple membrane enclosed vesicles termed apoptotic bodies (454). The

disassembled cell is finally phagocytosis by its neighbors. This form of cell death differs

from necrosis where cells typically lyse by loss of membrane (191). Apoptosis is part of

normal homeostatic mechanism whereby cells die during embryonic development, tissue

turnover and as a means of defense against pathogens. Metazoan development is

characterized by the over production of cells followed by apoptotic induction to cull the

excess cells at later stages of development such that the relative number of cells required

to achieve proper function is maintained. This phenomenon is first characterized in the

nematode Caenorhabditis elegans by Horvitz and others (468). Processes of assembly

and disassembly constantly maintain the normal human body's approximately 1014 cells.

It is therefore not surprising that inappropriate cell death (either too much or too little)

can give rise to disease conditions. Excessive apoptosis has been implicated in

neurodegenerative disease such as Alzheimer's (242, 387) and in tissue destruction that

occurs after vascular occlusions in the brain and heart (34, 62, 286). Similarly the lack of

apoptosis contributes to the development of tumors (2) and autoimmunity such as

autoimmune lymphoproliferative syndrome in humans (107, 339,439).









Apoptosis can be induced by a number of stimuli such as radiation, growth factor

depravation, receptor-mediated signaling and viral infections. There are basic pathways

by which a cell can be signaled to initiate apoptosis as shown in Figure 2. One pathway

is extrinsic, mediated via receptor-ligand interactions specifically the receptors related to

the tumor necrosis factor (TNF) and nerve growth factor (NGF) families including

TNFR1, TNFR2 and CD95/APO-1/FasR (147). These specialized cell surface receptors

are termed 'death receptors' (DR) (147). Ligand-receptor interaction results in receptor

oligomerization that causes specific cytoplasmic proteins to be recruited to the receptor

complexes. These cytoplasmic "adapter" proteins such as TRADD and FADD, then

interact with a group of cellular proteases called caspases. The activation of caspases

leads to the breakdown of cellular homeostasis that culminates in cell death.

Alternatively the second or intrinsic signaling pathway involves mitochondrial

dysfunction (Fig. 2). External cues such as loss of growth factor signals converge with

internal cues such as DNA damage and trigger the permeablization of the outer

mitochondrial membrane and leakage of mitochondrial pro-apoptotic effectors into the

cytoplasm (147). In addition the cytotoxic granule, granzyme B, a serine protease

produced by CTLs and natural killer (NK) cells has been shown to directly activate

caspases as well as the cytoplasmic protein Bid which activates the mitochondrial

pathway (26, 184). The pro-apoptotic stimulus results in the release of an array of

molecules from mitochondrial membranes, which ultimately cause cellular demise that

can be dependent or independent of caspase activation. The activation of caspases is by

far the main biochemical incident responsible for programmed cell death. Thus signaling

through DRs or the disruption of mitochondrial integrity results in the induction of


























Figure 2. Apoptosis. The induction of apoptosis can be signaled extrinsically involving
TNF receptors or Fas. Receptor complexes aid in the activation of initiator caspases such
as caspase-8 and -10 which in a cascade fashion ultimately in results in the activation of
terminal caspases such as caspase-3, -6 and -7. Cellular substrates that are involved in
DNA repair and homeostasis form targets for activated caspases. Apoptotic signals can
also originate from within the cell when mitochondrial integrity is compromised. Factors
normally residing within mitochondrial membranes such as cytochrome c then leak into
the cytosol and can cause activation of caspases. Mitochondrial proteins like AIF and
EndoG can induce apoptosis independent of caspase activation. Granzyme B can directly
activate caspases or can cleave cytosolic protein Bid. Cleaved Bid translocates to the
mitochondria and associates with Bax. Transcription factor p53 regulates bcl and bax
levels. Upregulation of bcl favors cell survival, whereas upregulation of bax favors cell
death. Abbreviations: Apaf-1, apoptosis associated factor-1; AIF, apoptosis inducing
factor; CASP, caspase; Cyt c, cytochrome c; DFF, DNA fragmentation factor; DNA-PK,
DNA protein kinase; FADD, Fas associated death domain; EndoG, endonuclease G;
TNFr, tumor necrosis factor receptor; TRADD, TNFR associated death domain; UV,
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apoptosis. Finally, neighboring cells and/or macrophages circumvent an inflammatory

response by phagocytosing the vesicles or apoptotic bodies formed as a result of

apoptotic induction.



Caspases

Caspases are defined as cysteine proteases that cleave protein substrates after

specific aspartic acid or the P1 residue (15). There are more than 42 mammalian

caspases described to date (217). Of these 12 are found in humans, caspases-1 to -10

(119), caspase-12 (105) and caspase-14 (91). There are also caspases cloned from

diverse organisms such as worms (459, 460, 468), insects (110, 168,365, 380) and hydra

(66).

Caspases are thiol proteases in that they make use of a cysteine side chain

nucleophile to hydrolyze cleavage of the peptide bond. Like other proteases they are

produced as zymogens and maturation involves cleavage of the propeptide after certain

aspartate residues. Caspase precursors contain an N-terminal prodomain that can vary in

length from 23 to 219 amino acids followed by regions encoding a large (p20) and a

small (pl 0) subunit. Sometimes spacer regions can also be present between subunits that

are excised during the maturation process (90).

The association of one large and one small subunit (p20/pl0 heterodimer) gives

rise to the catalytic domain. Each active enzyme is a tetramer composed of two such

heterodimers. The quaternary structure of caspases is unique among proteases and is

described as a homodimer of heterodimers containing two active sites at opposite ends.

While the requirement for an aspartic acid residue at the P1 position of the substrate is an









absolute, the two residues (P2 and P3) towards the amino side of P1 are not absolute but

are important in having an impact on substrate cleavage. The P4 residue on the other

hand appears to be important in determining substrate specificity. Caspases are activated

in a sequential manner beginning with those that initiate the cascade and ending in the

activation of effector caspases that result in the cleavage of the so-called death substrates.

By nature of their cleavage specificity, most caspases are capable of auto processing.

The length and presence of certain motifs on caspase prodomains are related to

their function. Caspases with large prodomains are typically involved in the initiation of

the apoptotic cascade. The death effector domain (DED) found in caspases -8 and -10

interacts with the DED of signaling adapter proteins that are associated with death

receptors involved in extrinsic signaling. Another motif, the caspase recruitment domain

(CARD) found in caspases -1, -2, -4 and -9 functions in promoting interactions between

one another and with adapter molecules. Short prodomain containing caspases are

typically effector caspases and are usually activated by initiator caspases.

Based on substrate specificities, caspases have been divided into three major

groups (119). Group I caspases are called inflammatory caspases since they function to

activate proinflammatory cytokines and include caspases-1, -4, -5, -11 and -12.

Caspases belonging to Group III function as initiator caspases and include caspases -2, -

8, -9, and -10. The initiator caspases cleave and activate Group II effector caspases that

include caspases-3, -6 and -7. Based on optimal substrate sequence specificity, caspase-2

has been placed in group II and similarly caspase-6 has been placed in group III (119).

The dual role of caspases in mediating apoptosis and inflammation indicates that these









two mechanisms are linked and underlines the importance of caspases in the regulation of

these two cellular processes.



Inflammatory Caspases-1, -4, -5, -11 and 12

Caspase-1 is the prototypical member of this group and was the first caspase to be

characterized. It was originally described as the interleukin-1 3 converting enzyme or

ICE (58, 413). In addition to processing proIL-1 to maturation, caspase-1 has also been

shown to process prolL-18 to the mature cytokine (122, 133). The human caspase-1

precursor is a 45 kDa polypeptide that is processed at 4 distinct sites. Initial cleavage

generates the 11.5 kDa prodomain while successive cleavages generate the large and

small subunits (413). The prodomain appears to be required for dimerization,

autoactivation (430) and activation of caspases-3 and -8 (404). Over expression of

caspase-1 has been linked to apoptotic induction in rat fibroblasts, mammalian COS cells

and neuronal cell lines (117, 134, 260). The first caspase knockout mice created was

caspase-1 deficient mice (209, 225). The importance of caspase-1 in the maturation of

IL- 1 3 and IL-18 is seen in the ability of caspase inhibitors to block the formation of

bioactive cytokines (194). Although mice deficient for caspase-1 are defective in

generating mature cytokines, the animals developed normally (209). There are also

reports that caspase- 1' -- mice were able to release biologically active IL- 13 in response to

subcutaneous injection of turpentine indicating the presence of alternate pathways for

prolL- 13 processing (100). Nevertheless the importance of caspase- 1 in inflammation is

supported by evidence showing that blocking caspase-1 activity or deleting the gene

rescues mice from lethal endotoxic shock (132). Caspase-1 knockout mice were also less








susceptible to hypoxic-ischemic brain injury suggesting the involvement of caspase-1 in

neuronal apoptosis (113,230). While the involvement of caspase-1 in processing

proinflammatory substrates is established, the activation of caspase-1 is less well

understood.

There is evidence to show that caspase-11 is an upstream regulator and is

responsible for the activation ofcaspase-1 (441). Although caspase-11 is a murine

caspase, it is thought to be a homologue of human caspase-4 (60% identical) (187). Like

caspase-1, caspase-11 can be induced by lipopolysaccharide (LPS) (440). Caspase-11

knockout mice are defective in processing prolL-103 (441) and apoptotic induction (187).

There is some evidence showing the activation of caspse- 11 by cathepsin B (356). There

is very little information known about the functions of caspase-4 and -5 (101, 186). Like

caspase- 1, they have been shown to process prolL- 1 3 (186) and prolL- 18 (122).

Caspase-5 can be induced by IFNy and LPS (228) and is found to be involved in receptor

mediated apoptosis (207). Of interest is the observation that caspase-5 can cleave the cell

cycle regulator Max at the unusual cleavage site of glutamic acid (207).

Caspase-12 was first identified in rodents and found to be localized in the

endoplasmic reticulum (ER) where it is activated by excessive stress to the ER (465).

Caspase-12 has been linked to neurodegenerative disorders (205) and was shown to

activate caspase-9 (266). Experimental evidence suggests that ischemic injury may

induce the activation of caspase-12 mediated by calpain (a cysteine endopeptidase

activated by intracellular calcium) resulting in apoptosis (284). It has been demonstrated

that procaspase-12 interacts with TNF receptor associated factor 2 (TRAF2) and is

activated in response to ER stress (465). A recent report suggests that catalytic









functionality of human caspase-12 has been lost since it is reported to contain a number

of mutations that prevent it from being expressed as a full length protein (105).



Apoptotic Initiator Caspases-2, -8, -9, and -10

The first caspase related disease condition described in humans is autoimmune

lymphoproliferative syndrome (ALPS) type II, an autoimmune disorder linked to

mutations in the initiator caspase-10 (439). Initiator caspases represent members

involved in both pathways leading to caspase activation namely the receptor mediated

extrinsic pathway involving caspase-8 and the intrinsic mitochondrial pathway involving

caspase-9. DR induced apoptosis involves receptor ligand interactions that aggregate and

act as scaffolds for caspase activation often termed the death inducing signaling complex

(DISC) (90). The recruitment of initiator caspases to the DISC involves the DEDs in the

prodomains of caspase-8 and -10 that are involved in binding DEDs on adaptor

molecules such as FADD in the DISC (438). Similarly the CARD found in caspase-2

and -9 prodomains has been shown to associate with CARDs on adaptor molecules such

as RAIDD facilitating the recruitment of caspase-2 to the DISC (5, 88). The induced

proximity model proposes that presence of high local concentrations of procaspase-8 in

the DISC is sufficient to stimulate the autoactivation of caspase-8 (283). This

phenomenon of autoactivation has been noted for caspase-9 and -10 (382, 438). An

alternative hypothesis is that conformational changes induced by binding adaptor

molecules can cause activation of proinitiator caspases (147). Activated caspase-8 can

either cleave the terminal effector caspases-3 and -7 directly or can cleave the









cytoplasmic protein Bid, which in turn activates the mitochondrial pathway (223).

Similarly activated caspase-10 has been shown to cleave procaspases-3, -7 and -8 (103).

Apoptosis can also be triggered without engaging the DRs, but instead causing

mitochondria to release proapoptotic molecules into the cytoplasm. One of these is

cytochrome c, a protein involved in electron transport, that now accumulates in the

cytoplasm and binds the apoptotic protease activating factor -1 (Apaf-1), a cytoplasmic

protein that shares homology with the C. elegans ced-4 apoptosis regulator. Cytochrome

c stimulates the formation of the mitochondrial apoptosome consisting of cytochrome c,

Apaf-1 and caspase-9. The binding of procaspase-9 to oligomers of Apaf-1 occurs

through the CARD regions of these two proteins. Like caspase-8, caspase-9 also

undergoes autoactivation induced by proximity of high concentrations of the proenzymes.

Caspase-9 processes the effector caspases-3 and -7 directly and has been shown to be the

apical caspase activated in the mitochondrial pathway responsible for the activation of

caspases-2, -6, -8 and -10 (374). Convergence of the DR and mitochondrial pathways to

cell death occurs as a result of caspase-8 mediated cleavage of cytoplasmic protein Bid,

which facilitates the release of cytochrome c from the mitochondria. This is probably as

a result of a feedback amplification loop since caspase-3 mediates activation of caspases-

2, -6, -8 and -10 following activation by caspase-9 (374).



Apoptotic Effector Caspases-3, -6 and -7

Caspase-3 which was first discovered as a gene product with high homology to

the C. elegans CED-3 protein and which caused apoptosis when overexpressed in Sf-9

insect cells (104). Subsequent work showed caspase-3 to be necessary for the cleavage








of poly (ADP-ribose) polymerase (PARP) an enzyme involved in DNA repair that is

proteolytically cleaved at the onset of apoptosis (289, 407). Caspase-3 is considered to

be the prototypic member of the effector caspases that function as the final executioners

of the apoptotic process (90). While the effector caspases can autoactivate, it has been

suggested that upstream caspases serve as the main activators. The initiator caspases-8, -

9 and -10 have all been found to cleave and activate procaspases-3 and -7 (90), while

caspase-3 has been shown to activate caspase-6 suggesting that this protease acts

downstream of caspase-3 (148). Caspase-3 can also be activated by caspase-1 (407),

caspase-4 (185), and caspase- 11 (187). The involvement of cytotoxic T cell mediated

apoptotic induction is demonstrated in the ability of the serine protease granzyme B to

directly activate caspses-3, -6 and -7 (103, 299).

Effector caspases target substrates that are essential in DNA repair pathways,

maintaining homeostasis and structural elements of cells. In the DNA repair pathway

targeted proteins include PARP, cleaved by all effector caspases, the catalytic subunit of

DNA dependent protein kinase (DNA-PKes) and the 70 kDa protein component of Ul

ribonucleoprotein (U 1-70 kDa) that are cleaved by caspse-3 (56). The characteristic

DNA ladder formation seen in isolated apoptotic nuclei can be induced by caspase-3,

which is known to directly activate caspase activated deoxyribonucease (CAD)/ DNA

fragmentation factor (DFF) (231) and also by caspse-7 (300). In addition, other

substrates include DNA topoisomerase I & II, RNA polymerase I upstream binding factor

(UBF) (57)

The induction of apoptosis gives rise to the cleavage of molecules that affect at

least three protein kinase pathways. Caspase-3 can activate the stress activated protein








kinase/Jun N-terminal kinase (SAPK/JNK) pathway which has been shown to up regulate

proapoptotic genes (90) (455) as well as activate protein kinase C 6 (PKC8) during

apoptosis (123). Activated cell cycle regulators such as cyclin dependent kinases (cdk)

have also been observed to be in apoptotic cells (473). The arrays of effector caspase

substrates include those that facilitate the breakdown of cellular structure. The nuclear

matrix proteins lamin A, B & C are cleaved by caspase-6 (347). All three effectors

cleave the structural nuclear mitotic apparatus protein (NuMA) (148). Cytoskeleton

proteins that are cleaved include actin, gelsoin, fodrin and keratin (373).



Intrinsic Activation of Caspases: Mitochondrial Permeability Transition Pore

The role of mitochondria in apoptosis was established following the discovery of

several mitochondrial proteins, which normally reside in the intermembrane space, but

are released into the cytoplasm following appropriate stimuli. Mitochondria have been

known for some time to be the energy centers of eukaryotic cells. Mitochondrial

transmembrane potential (Aym) represents the potential between ions distributed on either

side of the inner mitochondrial membrane that results in a chemical (pH) and electric

gradient needed for mitochondrial function (20). During the induction of apoptosis, Ay.m

collapse has been found to precede the changes associated with nuclear breakdown in a

number of cell types (208). Although the events leading to Arn disruption and

mitochondrial damage have not yet been elucidated, it is clear that pro- and anti-apoptotic

members of the B-cell lymphoma-2 (Bcl-2) family regulate the intrinsic mitochondrial

pathway.









The Bcl-2 family consists of more than 12 proteins that have been divided into

three groups based on structural and functional similarities (2, 17). Members of the anti-

apoptotic first group including Bcl-2 and Bcl-XL, contain four conserved Bcl-2 homology

(BH) domains (BH1-BH4). The C-terminal hydrophobic tails localize the proteins to the

outer mitochondrial surface while the major portion of these proteins face the cytoplasm.

The second group consists ofpro-apoptotic members including Bax and Bak,

characterized by three BH domains and the C-terminal hydrophobic tail. Members of the

third group are diverse and pro-apoptotic including Bid and Bik, containing only the BH3

domain. Bcl-2 family members can form homodimers, but more importantly the pro- and

anti-apoptotic members interact to form heterodimers. Large numbers of such

heterodimers can be found in the cell. Ultimately the levels of pro- versus anti-apoptotic

family members determine the fate of the cell. An excess ofpro-apoptotic Bcl-2 family

members causes cell death conversely the presence of larger numbers of anti-apoptotic

members is protective.

There are a number of models to explain the mechanism of AYm disruption

mediated by the formation of permeability transition pores (PT) that allow the dissipation

of inner membrane ion gradients (147). Since Bcl-2 members oligomerize, they could

potentially form channels to facilitate protein transport. Alternatively Bcl-2 family

members could interact with other proteins to form channels. The voltage dependent

anion channel (VDAC) can bind several Bcl-2 members that regulate its activity (147).

Similarly the adenine nucleotide translocator protein (ANT) has also been implicated to

play a role in Bax induced apoptosis (244). And finally it has been proposed that Bcl-2

members themselves induce the rupture of mitochondrial membranes thereby disrupting









Aym and facilitating the release of mitochondrial pro-apoptotic proteins into the

cytoplasm.

The release of cytochrome c leads to formation of the mitochondrial apoptosome

in conjunction with cytoplasmic Apaf-1 and procaspase-9, thus activating caspase-9.

Second mitochondrial activator of caspases (SMAC) or direct IAP binding protein with

low pI (Diablo), is a 25 kDa pro-apoptotic polypeptide released from mitochondria (87,

434). The first four amino acids of mature SMAC/Diablo, Ala-Val-Pro-Ile (AVPI) binds

baculovirus inhibitor of apoptosis repeat (BIR) 3 domain of X chromosome encoded

inhibitor of apoptosis protein (XIAP) (59). SMAC/Diablo can also bind the BIR2

domain of XIAP, a region important for XIAP mediated inactivation of caspases-3 and -

7. Thus SMAC binding to XIAP releases active caspases-3, -7 and -9 thereby promoting

cell death. The serine protease Htra2/Omi is also released from mitochondria and has

been shown to interact with XIAP and relieve inactivation of caspases (394). Apoptosis

inducing factor (AIF) and endonuclease G (EndoG) have recently been implicated in cell

death independent of caspase activation (224, 309, 393). Thus as a result of

mitochondrial dysfunction cell death occurs that is both dependent and independent of

caspase activity.



Extrinsic Activation of Caspases: Death Receptor Mediated Signaling

Apoptotic death, an essential feature of the mammalian immune system, regulates

lymphocyte maturation, receptor repertoire selection, homeostasis and cell-mediated

immune response. This pathway of programmed cell death makes use of specialized cell

surface receptors termed the death receptors (DRs) that belong to the tumor necrosis









factor receptor (TNF-R) superfamily. Members of the DR family include Fas/CD95,

TNF-R1, DR3, DR4 and DR5 (188). The common feature of these molecules is the

presence of the cytoplasmic death domain (DD) that is essential for apoptotic signal

transduction. Receptor binding leads to oligomerization and formation of the death

inducing signaling complex (DISC) that allows the recruitment of intracellular adapter

proteins to the DISC.

Interaction between the death domains of the receptors and adapter proteins such

as Fas associated death domain protein (FADD) and TNF-R associated death domain

protein (TRADD) are responsible for the binding. The adaptor molecules also contain

the death effector domains (DED) that allow recruitment of procaspases-8 and -10 to the

DISC. The initiator caspases are autocatalyzed and serve as activators of the terminal

effector caspases that cleave the death substrates. The importance of adapter molecules

like FADD in apoptosis is evidenced in cell lines derived from FADD knockout mice that

fail to undergo apoptosis induced by CD95 ligation. However FADD"-' fibroblasts were

still sensitive to cell death induced by drugs that utilize the mitochondrial pathway (470).

The discovery of viral and cellular proteins that function to block the signal

transduction events downstream of the DISC gave rise to a new family of anti-apoptotic

proteins termed the FLICE inhibitory proteins (FLIPs). The viral FLIP found in certain

herpesviruses and poxviruses inhibit the recruitment of procaspase-8 to the DISC

underlining the importance of this pathway in pathogen clearance. Casper/cFLIP is the

cellular homolog of vFLIP and contains two DEDs and a defective caspase-like domain.

Casper can associate with the DEDs of FADD and caspase-8 thereby interfering with the

formation of a functional DISC. As expected cells from Casper knockout mice are highly









sensitive to receptor mediated apoptotic induction (462). Interestingly gene disruptions

in FADD"', Casper'-, and caspase-8"' cause embryonic lethality at similar stages and

display abnormalities in heart development (432, 462, 463).

Diseases evidence the importance of DR mediated cell death in the regulation of

the immune system where either too little or too much apoptosis occurs. The

lymphoproliferation mutation (lprc) in mice is a point mutation in the CD95 DD that

abolishes apoptotic signal transduction causing symptoms similar to those seen in

systemic lupus erythematosus. Very similar symptoms are seen in generalized

lymphoproliferative disease (gid) mice where a point mutation at the carboxy terminus of

the CD95 ligand blocks ability to ligate the CD95 receptor. In both cases there is an

accumulation of aberrant T cells. In humans, autoimmune lymphoproliferative syndrome

(ALPS) type I is a dysfunction of the CD95-CD95L system showing severe

autoimmunity with non-malignant lymphadenopathy and an increase in T cells. Thus the

failure to purge self-reactive lymphocytes can cause autoimmunity. AIDS is

characterized by a depletion of CD4+ T helper cells in the peripheral blood of patients

with human immune deficiency virus (HIV). Viral regulatory proteins such as HIV-1 Tat

enter non-infected cells and causes them to be hypersensitive to apoptosis by CD95

ligation. The binding of HIV gp 120 to CD4 also increases CD4 T cells to undergo

apoptosis. Thus receptor-mediated apoptosis seems to be a mode of CD4+ T cell death

that plays a role in the immune regulation of HIV infection.









Granzyme B

Cytotoxic T cells CTLs and NK cells can selectively induce apoptosis in by

binding the death receptor CD95 on target cells or alternatively can cause apoptosis via

the cytotoxic granule granzyme B (GrB). GrB, a 35 kDa protein, is a member of the

granzyme family of lymphocyte serine proteases and is the only enzyme known to date

that shares aspartate cleaving specificity with caspases. Granzymes in general are

involved in cytolysis brought about by CTLs and NK cells. There are 5 granzymes

known to date in humans -A, -B, -H, -K and -M (184) the most abundant being GrA and

GrB (320). Granzymes are able to enter the target cells via the pore forming protein

performin. Performin shares structural and functional homology with the complement

membrane attack complex and gets inserted into target cell membranes where it

polymerizes to form perforin pores. Granzymes are synthesized as pre-zymogen

peptides. The pre or leader sequence targets the molecule to the secretary pathway.

Cleavage of the propeptide activates the enzymes. While purified granzymes alone have

no cytotoxic effects, their enzymatic substrates are potent cytolytic agents. GrB has been

shown to cleave procaspases-3, -6, -7, -8, -9, and -10 thus activating both initiator and

effector caspases. Cleavage of the Bcl-2 family member Bid directly by GrB activates

the mitochondrial pathway in apoptosis. In addition GrB has been shown to cleave death

substrates including PARP, DNA-PKcs, and NuMA (16, 114). Thus GrB plays an

important role in the induction of apoptosis through both the intrinsic and extrinsic

pathways.









Apoptosis in Avian Species

Apoptosis in the avian system is less well understood compared to the information

available from the mammalian system. Both mitochondrial mediated and DR mediated

pathways of apoptotic induction have been observed in birds. Most of these studies have

been done in chicken, quail and duck. A number of avian death receptors have been

identified to date. The first avian death receptor protein TVB was identified as the

receptor for avian leucosis sarcoma viruses (49). TVB is 37% identical to human DR-5

death receptor and has been shown to function via its death domain (48, 49). The chicken

B6 (chB6) molecule, widely used as a B lymphocyte marker, does not contain any

apparent DDs, but has been shown to induce apoptosis when cross-linked with antibodies

or overexpressed in murine cells (115, 317). The chicken homolog to human DR-6 is

76% identical to its human counterpart and contains a death domain and two TRAF

binding sites (45). Elevated levels of DR-6 protein correlated with apoptotic induction in

the hen ovary (45). Characterization of partial cDNA sequences of chicken Fas and TNF-

RI have shown 37% and 42% identity to the human homologs respectively (46).

Members of the bcl2 family have also been identified in chickens. The chicken bcl-xl

homolog functions to protect hen granulosa cells from apotosis in the phosphorylated

state (177). The bcl-2 homolog Nr-13 was originally identified as an antiapoptotic

protein activated upon infection by Rous sarcoma virus (124). Purified Nr-13 is able to

bind cytochrome c with high affinity suggesting that it blocks the formation of the

mitochondrial apoptosome involving cytochrome c and Apaf-1 thereby regulating the

induction of apoptosis (265). An IAP protein has also been identified in chickens.









Inhibitor of T-cell apoptosis (ITA) protein contains BIR domains as well as the zinc

finger motif characteristic of IAPs (176).

To date four avian caspases have been cloned and characterized. Chicken

caspases-1, -2, -3 and -6 (174, 175, 178). There are no reports to date of purified chicken

caspases showing biochemical activity. Chicken caspase-1 while being 44% identical to

human caspase-1 has two cysteine residues (QCCRG) in the active site pentapeptide

compared to its human homolog (QACRG) that has only one. The chicken homolog of

caspase-2 is 70% identical to its human counterpart and contains the conserved QACRG

catalytic site sequence. Chicken caspase-3 and -6-like activity was noted in granulose

cells treated with the apoptotic inducer okadaic acid. Both chicken caspase-3 and -6 are

>65% identical to the human enzymes with complete conservation in the active site

region. Caspase-6 gene disruptions in a chicken cell line had no effect on the induction

of apoptosis (347). Chicken caspase-6-like its mammalian counterpart was seen to

cleave lamins A and C. Available reports of apoptosis in the avian system suggest

conservation of both DR and mitochondrial pathways. The identification of novel

molecules in the future will highlight differences and help gain a better understanding of

this process in birds.



Viral Inhibitors of Apoptosis

Viruses are obligate intracellular parasites and therefore induction of an apoptotic

reaction to infection is a highly effective host innate response that controls and limits the

spread of the invading pathogen. Viruses, in order to survive have developed multiple

ways to either neutralize or delay the cell death machinery thereby maximizing the








production of progeny particles. Virus infection itself generates proapoptotic signals.

Adsorption of non-infectious virus particles to the cell surface can trigger apoptotic

signaling. Avian leukosis virus enters cells via the TVB receptor, (a member of the DR

family with significant homology to human DR-5) which has been shown to transduce

apoptotic signal (48). Viral mechanisms that block both the extrinsic and intrinsic

pathways of apoptosis have been identified. The mitochondrial pathway can also be

triggered following virus infection. Viral replication cycle activates cell cycle regulators

such as p53 is thought to involve the Bcl-2 family of proteins to induce mitochondrial

dysfunction (131). Respiratory syncitial virus can activate the ER resident caspase 12

(37). The identification of viral inhibitors in both pathways of programmed cell death

indicates the importance of apoptosis in controlling a viral infection. Inhibitors of both

the intrinsic and extrinsic pathways are discussed.



Viral inhibitors of Mitochondria: PT pore modulators and Bcl-2 homologs

Recently vaccinia virus was shown to inhibit apoptotic induction mediated by

mitochondria (444). Infections of Jurkat cells with vaccinia virus deleted for caspase

inhibitor SPI-2 were found to be resistant to Fas-induced cell death, prevent the loss of

mitochondrial membrane potential and decrease cytochrome c levels as well as caspse-3

activity. The mechanism of vaccinia virus inhibition of mitochondrion mediated

apoptosis is not clearly understood but is believed to involve the ability of the virus to

modulate the permeability transition pore. The Hepatitis B virus HBx protein binds

human VDAC and inhibits apoptotic induction by altering transmembrane potential

(324). Viral mitochondria localized inhibitor of apoptosis (vMIA) encoded by









cytomegalovirus forms a complex with ANT and blocks permeablization of the

mitochondrial outer membrane (128).

Modulations of mitochondrial apoptotic pathways have been observed in a

number of other viruses. Epstein-Barr virus, a y herpesvirus encodes two bcl-2

homologs. The BALF-1 protein contains two BH domains and protects transfected HeLa

cells from undergoing apoptosis when induced with anti-Fas antibody (241). The

mechanism of BALF- 1 function seems to be via association with proapoptotic bcl-2

members, Bax and Bak. The BHRF-1 protein is less similar to bcl-2 and bcl-xl than

BALF-1 but has shown to protect human B cell lines from apoptotic induction with

ionomycin (146, 241). The adenovirus E1B19K protein is also a bcl-2 homolog that

blocks apoptosis during virus infection (327). E 1B19K does not block the activation of

caspase-8 but rather interacts with Bax causing inhibition of cytochrome c release from

mitochondria and subsequent activation of caspase-9 (313). Further characterization has

shown it to specifically block oligomerization of Bax by preventing exposure of the Bax

BH2 epitope (391,392).



vFLIPs: Inhibitors of DISC

Collectively, proteins that inhibit the activation of initiator caspases in the DISC

belong to the family of FLICE (caspase-8) inhibitory proteins (FLIPs). A common

characteristic of FLIPs is the presence of DEDs. DED containing proteins have been

located by database searches of herpes and poxvirus genomes (32). The equine herpes

virus E8 protein interacts with the prodomain of caspase-8 preventing its activation and

possibly its recruitment to the DISC. Ectopic expression of E8 protects HeLa cells from









TNFRI and Fas induced apoptosis (32). MC159 ofMolluscum contagiosm virus contains

two DEDs, binds both caspase-8 and FADD and blocks apoptosis induced by Fas, TNF

and TRAIL (120). Similar vFLIPs have been found in a number of herpes viruses some

of which associate with kinases of the I,:B kinase complex (127, 229).



Baculovirus Caspase Inhibitor: P35

The baculovirus anti-apoptotic P35 proteins are unique in that they do not share

homologies with any other known proteins. Interestingly these proteins share no

homology to poxvirus serpins, yet they inhibit similar target proteinases. P35 was first

identified in the insect virus Autographa californica nuclear polyhedrosis virus (AcNPV)

where P35 mutants induce apoptosis in the Sf-21 insect cell line (isolated from

Spodopterafrugipedra) (68). P35 proteins from a number ofbaculoviruses show distinct

abilities to inhibit apoptosis (267). Most of the P35 functional data generated to date

have used AcNPV P35. In vitro studies have implicated P35 to be a suicide substrate for

caspases (4). P35 has been shown to inhibit all three groups of mammalian caspases and

specifically caspases-1, -3, -6, -7, -8 and -10 (474). In addition P35 has also been shown

to inhibit the C.elgans CED-3, Sfcaspase-1 as well as gingipain (4, 378, 459). Recently,

the crystal structures of P35 with caspase-8 an apical initiator caspase and also with

effector Sf caspase-1 I have revealed clues to its function mechanisms (92, 106, 458). The

reactive site loop (RCL) of P35 associates with the molecule's main P3 sheet core such

that the Asp residue required for protease recognition is exposed at the apex of the loop.

Following cleavage of the RCL, there forms a covalent thio-ester linkage between the

target proteinase and P35. Cleavage also induces a dramatic conformational change in









P35 where the N-terminal of P35 gets repositioned into the active site of the caspase

protecting the thio-ester linkage from hydrolysis. Interestingly the presence of certain

motifs in effector caspases but absent in initiator caspases explains the preference of P35

to complex with terminal rather than apical caspases. Ectopic expression of P35 has

shown it inhibits apoptosis in a number of systems including insects, nematodes and

mammalian cells where the apoptotic signals tested included virus infection, induction by

actinomycin D, induction by Fas and TNF, NGF depravation, developmentally

programmed cell death and induction by X-radiation (259). In each case, P35 was able to

either block completely or significantly delay apoptosis. In the light of these studies, the

broad range of activity ascribed to P35 is suggestive that the molecule is a pan-caspase

inhibitor interrupting the activity of a highly conserved and ubiquitous component of the

death machinery.



Baculovirus Inhibitor of Apoptosis Proteins (lAP)

The family of inhibitor of apoptosis (lAP) was first described in baculoviruses by

Lois Miller and others (75). lAPs have also been identified in yeast, worms, insects and

mammals (350). Two zinc-binding motifs are characteristic of lAPs, the first is the

baculovirus IAP repeat (BIR) and the second being a RING domain. The BIR motif can

be found in multiple copies in a given lAP, while the RING domain is typically a single

copy located at the carboxy terminal end of the molecule (350). The RING domains

function to catalyze the degradation of IAPs and target protein through ubiquitylation

processes. The BIR domains seem to be essential for lAPs to function as anti-apoptotic

agents (89). Baculovirus lAPs were discovered by virtue of their ability to block









apoptosis in Sfcells infected with a AcNPV P35 mutant (80, 337). The CplAP and

OpIAP proteins (isolated from baculoviruses Cydia pomonella granulosisvirus and

Orgyia pseudotsugata nucleopolyhedrovirus respectively) can also inhibit apoptosis

induced by a variety of signals including induction by, actinomycin D, expression of the

reaper and Doom genes of Drosophila melanogaster and expression of ICE in a

mammalian system (153). The baculovirus IAPs mode of inhibiting the Sfcaspase-l

activity is thought to affect the activation of this insect caspase, implying that the

mechanism by which IAPs function is different from the P35 mode of action (365). A

number of lAP homologues have been found in insects and mammals including the

human neuronal apoptosis inhibitory protein (NAIP), cIAP-l, cIAP-2 and XIAP (350).

The mechanism by which viral lAPs act in inhibiting apoptosis is not fully understood.

Studies with the crystal structures of human XIAP has elucidated a possible mechanism

of action that could be similar for all lAPs. The BIR3 domain of XIAP is thought to aid

the cleavage of caspase-9 and directly bind the small subunit at the C-terminus of

caspase-9 inactivating it (383, 390). With respect to caspase-3 and -7, XIAP seems to

have a completely different mode of inhibition than that seen with caspae-9 (60, 164,

338). In this case BIR2 domain is involved and complexes with caspase-3 and -7. A

linker domain N-terminus to BIR2 functions to block the substrate groove in the caspase,

in a backward or opposite orientation that is different from substrate recognition

sequences making it a novel mechanism for caspase inhibition. XIAP is not a substrate

for caspase-3 or -7 but rather functions to mask the active site. It will be of interest to

know whether the viral IAPs also function in a similar manner.









Caspase Independent Induction of Apoptosis

Apoptosis in the absence of active caspases seems to represent a pathway

conserved in slime mold (Dictyostelium), worms (C.elegans) and mammals (234, 297).

Two proteins have been identified to date that are released from mitochondria and are

postulated to play a role in apoptosis independent of caspase activation. The first is a

novel mitochondrial cell death inducer called the apoptosis inducing factor (AIF) (393).

Mammalian AIF is a 57 kDa flavoprotein that shares homology to oxidoreductases from

vertebrates (Xenopus laevis), non-vertebrates (C.elegans), plants, fungi and bacteria

(234). It was originally discovered as the factor able to induce caspase independent

chromatin condensation and DNA fragmentation in isolated nuclei, but itself has not been

seen to exhibit DNAse activity (393). AIF can also cause permeablization of

mitochondrial membranes and induce release of cytochrome c suggesting a role in a

positive feedback loop with the apoptosome pathway (393). Deletion of AIF in mice

causes embryonic lethality (182). While cells from knockout mice remain sensitive to

apoptosis in response to serum withdrawal, they are resistant to apoptosis induced by the

oxidative stress inducer menadione (182). Since AIF is also an oxidoreductase, it is

unclear whether observations seen in KO mice were due to loss of apoptotic activity

alone or due to the elimination of oxidoreductase activity as well.

The second toxic molecule implicated in caspase independent apoptosis is

endonuclease G (EndoG), which, like AIF, is also released from mitochondria (309).

EndoG is a 30 kDa nuclease, which was originally linked to mitochondrial DNA

replication based on its location and substrate specificity (74). Unlike CAD/DFF, EndoG

is able to induce nucleosomal DNA fragmentation independent of caspase activation









(224). It has also been implicated in DNA fragmentation seen in DFF deficient mouse

embryonic fibroblasts following induction of apoptosis by UV-radiation and TNF

treatment (469). A homolog to EndoG in C.elegans has been identified as CPS-6, which

shows similarity in sequence, localization and biochemical properties to mammalian

EndoG (309). Mutant C.elegans deficient in CPS-6 can be rescued by mouse EndoG

(309). The involvement of EndoG in cell death indicates the presence of an apoptotic

pathway, independent of caspase activation that is conserved from worms to mammals.

Although the caspase independent pathway is not completely understood, the

identification of AIF and EndoG clearly supports the existence of an apoptotic program

parallel to caspase activation.



Poxviruses

The Poxviridae are a family of large double stranded DNA viruses that replicate in the

cytoplasm of infected cells. The taxonomy of poxviruses is based on hosts they infect and

include the two subfamilies Chordopoxvirinae containing vertebrate viruses and

Entomopoxvirinae containing the insect poxviruses (Table 2) (271). The

Chorodopoxvirinae comprises eight genera: Orthopoxvirus, Parapoxvirus, Avipoxvirus,

Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. While

there are three genera within Entomopoxvirinae, these viruses may need to be reclassified

based on recent sequence information that suggests that they may not be as closely

related as previously thought (3, 27). Both variola virus (Orthopoxvirus genus) and

molluscum contagiosum virus (Molluscipoxvirus genus) are the only poxviruses to

exclusively infect humans (271).










Table 3. Taxonomy of poxviruses


Genera Members

Orthopoxvirus Cowpox, monkeypox, buffalopox,
vaccinia, Variola, rabbitpox,
camelpox, ectromelia, raccoonpox,
taterapox, volepox
Parapoxvirus Pseudocowpox, parapoxvirus of red
deer, bovine popular stomatitis virus,
~________ orf, paravaccinia virus
Vertebrate Avipoxvirus Canarypox, fowlpox, juncopox,
Vererat mynahpox, pigeonpox, quailpox,
poxviruses sparrowpox, starlingpox, turkeypox
Capripoxvirus Goatpox, sheeppox, lumpy skin
disease virus
Leporipoxvirus Myxoma virus, rabbit fibroma virus,
hare fibroma virus, squirrel fibroma
virus
Suipoxvirus Swinepox virus
Molluscipoxvirus Molluscum contagiosum virus
Yatapoxvirus tanapox virus, Yaba monkey tumor
virus
Entomopoxvirus A Anomala cuprea, Aphodius
tasmaniae, Demodema boranensis,
Dermolepida albohirtum, Figulus
____ subleavis, Melolontha melolontha
Entomopoxvirus B Amsacta moorei, Acrobasis zelleri,
Insect Arphia conspersa, Choristoneura
t biennis, Choristoneura conflict,
Poxviruses Melanoplus sanguinipes, Oedaleus
~____~___senigalensis, Schistocera gregaria
Entomopoxvirus C Aedes aegypti, Camptochironomus
tentans, Chironimus luridus,
Chironomus plumosus, Chironomus
attenuatus, Goeldichironomus
______________haloprasimus
California harbor sealpox, cotia
Unclassified virus, dolphinpox, embu virus, grey
Uncassified kangaroopox, marmosetpox,
poxviruses Molluscum-like poxvirus, Nile
~~~____~~______crocodilepox, mule deerpox virus


http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm









By far the most infamous poxvirus is variola, the causative agent of smallpox

infections in humans. Smallpox produced a systemic disease with a generalized rash in

humans and has been documented for about 2000 years (102). Of importance is that it is

the first and only human disease to date that has been purposefully eradicated. The

concept of variolation i.e. inoculation of dried smallpox scab material into the skin to

induce mild disease and subsequent immunity was practiced since the early 10th century

(102). It was not until 1798 when Edward Jenner demonstrated that inoculation with

cowpox could protect against smallpox. This new method, vaccination, quickly gained

widespread use and eventually led the eradication of smallpox in 1977 as a result of mass

vaccination programs carried out by the World Health Organization.



Poxvirus Lifecycle

Poxvirus genomes are linear double stranded DNA molecules that are covalently

joined at the termini. The internal core region of the genome is highly conserved among

poxviruses whereas variation occurs at the ends of the genome within the inverted

terminal repeat (ITR) regions. Genes located in this region are typically not essential for

viral growth in tissue culture, but contribute to viral pathogenesis in vivo. Poxviruses are

unique in that the entire life cycle is accomplished in the cytoplasm of the infected cell

(Figure 3). The receptor that mediates viral entry is unknown at present. However viral

entry is mediated by membrane fusion that is dependent on pH. Viral gene expression

occurs in three stages. The early genes are transcribed within the viral core that contains

all the transcription machinery. These include growth factors, immune defense

molecules such as CrmA and factors required for DNA replication. Intermediate genes










DNA


rEntry/uncoating


1
Early gene
expression


Intermediate /
transcription factors,
Immunomodulatory 0
proteins


f replication



Intermediate gene
expression

L |
gP Late gene expression
U ,


_j H f/1I _1E\V 1-1 barlyt
IEV_ @Str


EEV Golgi _____
-- Ggwrapping I j
IMV Immature
Particle Assembly


transcription factors
uctural proteins
Late enzymes




j


Figure 3. Poxvirus replication cycle. The cytoplasmic infection of a cell with an
infectious poxvirus particle. Abbreviations: EEV, extracellular enveloped virus; IMV,
intracellular mature virus; IEV, intracellular enveloped virus.


EEV


CEV


4" .









are transcribed concomitantly with DNA replication. Intermediate gene products are

factors for late gene transcription. Late gene products include structural proteins,

enzymes and early transcription factors. The viral DNA is then packaged within the

cytoplasmic viral factories and maturation proceeds to form infectious intracellular

mature virus (IMV) particles. IMV can be enveloped by membranes derived from the

golgi apparatus to form intracellular enveloped virus (IEV) which upon exiting the cell

looses a membrane layer and is termed extracellular enveloped virus (EEV).



Importance of Poxviruses

Poxviruses are extremely efficient in evading the effectors of antiviral immunity. Many

of these mechanisms target conserved cellular pathways. The knowledge gained from the

interactions of these viral proteins would be useful in understanding the immune response

to a poxvirus infection. Such studies also help in elucidating mechanisms of immune

evasion used by the virus. Therapeutic strategies based on models generated from viral

studies could be used to design molecules to modulate immune responses that occur in

other pathological conditions. Vaccinia virus is also an excellent vector for the

expression of foreign proteins, but the virus also elicits a tremendous immune response.

Therefore while the virus has potential as a vector in gene therapy, it might be

advantageous to minimize the side effects of treatment (immuneregulation) without

compromising gene delivery. Identifying and possibly modifying the genes responsible

for the vigorous immune response can improve its use in gene therapy. Finally, though

smallpox has officially been eradicated, interest in smallpox is sustained due to its

potential use as a weapon of mass destruction in biological warfare. Smallpox is also









similar to another related poxvirus namely monkeypox that causes zoonotic infections of

humans producing a clinically identical disease.

Poxviruses encode a number of molecules that aid in subverting and blocking the

host immune response at various levels. As described earlier, poxviral immune evasion

proteins include those that function to block complement activation, TNF, IL-103, IL-18,

IFNa/P signaling via either binding proteins such as the virally encoded IL- 18 binding

protein or receptor homologs such as CrmB-E which bind TNF. In order to modulate

immune cell trafficking poxviruses encode proteins that bind chemokines or are

chemokine homologs. Poxviruses encode intracellular molecules to block activation of

cellular responses to viral replication, which include the double stranded RNA binding

protein E3L and K3L a homolog to cellular initiation factor eIF-2cc. Also included

among the intracellular proteins believed to interact with the host immune system are

poxvirus serpins, which are members of the serine proteinase inhibitor family. The

subject of this dissertation work involves the serpin CrmA first identified in cowpox virus

and subsequently found to have homologs in all orthopoxviruses sequenced to date

including variola and vaccinia viruses. Orthopoxviruses encode a total of three serpins

SPI-1, SPI-2/CrmA and SPI-3, which will be discussed later.



Serine Protease Inhibitors (Serpins)

The serpin protein superfamily is characterized by a conserved structure and

mode of action that can be described as suicide substrate-like inhibition (368). Serpins

are ubiquitous in nature, found in animals, plants, certain viruses and more recently in

bacteria (368, 449). Based on phylogenetic analysis serpins are grouped into 16 clades









and 10 divergent orphans (368). Although most serpins function to inhibit serine

proteases, there exist examples of cross class inhibition such as that exhibited by the

poxviral serpin CrmA and the cellular PI-9 which can inhibit both serine and cysteine

proteases (e.g. caspases). In addition some serpins such as ovalbumin are non-inhibitory

while corticosteroid binding globulin (CBG) and thyroxine binding globulin (TBG)

function as transport proteins (165, 311). In mammals serpins are involved in regulating

a number of processes including coagulation, fibrinolysis, inflammation, complement

activation, organ development and extracellular homeostasis as shown in Figure 4.

A metastable structural conformation is extremely important for the function of inhibitory

serpins. The conserved secondary structure is composed of three P-sheets and at least 7

(typically 9) a-helicies. The exposed reactive center loop (RCL) is a region spanning

about 20 amino acids located at the C-terminus of the molecule which serves as the

proteinase recognition site as shown in Figure 5. Within the RCL lies the P 1 residue that

determines target proteinase specificity. The P1-PI' peptide bond is cleaved during

scissile bond formation with the proteinase. Initially the proteinase forms a non-covalent

Michaelis-like complex by interactions involving residues flanking the scissile bond.

Attack by the active site serine (in the case of serine proteases) on the P1 residue

leads to the formation of a covalent bond between the carbonyl of the P 1 residue and the

protease. At this point, the RCL begins to insert itself into 13-sheet A of the serpin while

still attached to the protease. The consequence of complete loop insertion is a 70A

translocation of the target proteinase that effectively distorts its active site resulting in

inactivation of the enzyme. The example of trypsin inactivation by alpha-1 antitrypsin is

shown in Figure 6. The formed acyl intermediate is stable from hours to weeks and is



















tumor cell invasion


prohormone conversion


SPCI (SERPINAS)
A Till (SERPINCI)
HCFII (SERPINDIJ
PAIl (SERPINEI)
ECM maintenance
and remodelling ---
I' PI(SERPINAI) PNI (SERPINE2)
alACT(SERPINA3) HSP47(SERPINHI)
PAll (SERPINEI) CBP2 (SERPINH2)


hormone transport

/CBG(SERPINA6) I
TBG (SERPINA7) PCI (SERPINAS)




inflammation &
"'- T complement activation
1 PI (SERPINAI)
adACr (SERPINA3)
SKAL(SERPINA4)
MNEI (SERPINBI)
oip-serins (SERPINB*)
blood pressure regulation
\' AGT(SERPINA8)
7- .....angiogenesis
PEDF (SSERPINFI)
maspiR (SERPINBS)?
A Till (SERPINCI)?
P411 (SERPIEI)?
T fibrinolysis


apoptosis
1 OlPI (SERPINAI)
-LIACT(SERPINA3)
av-.sepiins (SERPINB*)?


Figure 4. Serpins as physiological regulators. Serpins are involved in numerous
functions both as positive and negative regulators. From reference 368.
























P14....P4 P3 P2P1-P1' P2' P3' P4'......P14'
t
Scissile Bond




Figure 5. Serpin RCL. Position of the C-terminal reactive center loop (RCL) in the
serpin molecule. P 1 residue within RCL determines specificity for proteinase.











Trypsin (active)


Serpin-proteinase complex


Native alpha-1 antitrypsin


Trypsin (disrupted)


Figure 6. Inhibition of trypsin by alpha-1 antitrypsin. Following formation of the
serpin-proteinase complex, the scissile bond is cleaved (P1 '-P1). RCL insertion into the
backbone of alpha-1 antitrypsin (serpin) causes Trypsin (target proteinase) to be
translocated such that its active site is effectively distorted resulting in inactivation of the
enzyme. From Huntington, et al. Nature, 2000


MM+









eventually hydrolyzed releasing the trapped previously active enzyme and cleaved serpin.

Serpin-protease complexes involving serine proteases are SDS stable and can be readily

detected on reducing gels. Whereas complexes with cysteine proteases, such as with

caspases, are intrinsically unstable due the nature ofthiol ester linkages that are easily

hydrolyzed and can only be detected on native, non denaturing gels.



Poxvirus Serpins

The first poxvirus serpin discovered was CPV CrmA (cytokine response modifier A). A

total of three serpins SPI-1, SPI-2/CrmA and SPI-3 are present in orthopoxviruses. SPI-1

mutants of RPV display host range restriction. SPI-1 can form complexes with cathepsin

G (263) and more recent work suggests that SPI-1 may interact with the viral DNA

replication machinery (235). Disruption of the SPI-3 gene in VV or CPV results in cell

fusion and the formation of giant syncytium (422). SPI-3 expressed in vitro can form

stable complexes with some serine proteinases including trypsin, urokinase and tissue

plasminogen activator (420). Mutational analysis reveals that serpin function is not

required for SPI-3 to inhibit cell fusion during virus infection, suggesting SPI-3 to be a

bi-functional protein, with two separate functional motifs (423).

The SERP1, SERP2 and SERP3 proteins are serpins encoded by Myxoma virus

(135, 314, 428). SERP1 is the only secreted viral serpin and in vitro experiments have

shown SERP 1 to inhibit serine proteinases including human plasmin, urokinase and

tissue plasminogen activator, as well as C Is of the complement system suggesting an

anti-inflammatory activity for this serpin (442). The biochemical properties of SERP1

are similar to SPI-3 of orthopoxviruses. Indeed in vivo studies have proven the potent









action of SERP1 against inflammation (237). Although SERP1 and SPI-3 share similar

inhibitory profiles in vitro, SPI-3 cannot substitute for SERP 1 in myxoma virus infections

of rabbits (442). The SERP2 protein shares a 45% similarity with CrmA/SPI-2 from

Orthopoxvirus and is also functionally similar to CrmA/SPI-2. SERP2 will be discussed

in more detail later. SERP3 was recently identified from myxoma virus and despite

having significant sequence deletions, has been proposed to retain overall serpin fold

(135). Deletion of SERP3 from myxoma virus causes attenuation of disease and prevents

viral spread to secondary sites in rabbits, suggesting a role in virulence. It is not yet

known if this serpin can inhibit proteases.

Little is known about the swine poxvirus (SPV) serpin SPI-7 that contains an Asp

at P1 like CrmA/SPI-2. SPI-7 does not reveal any activity against caspases tested to date,

although SPV mutants deleted for SPI-7 have shown some attenuation (Pierre Mussy &

Richard Moyer unpublished). There are 5 serpins found in the fowlpoxvirus genome, in

addition serpins have been identified in Yaba-like disease virus (YLDV) and lumpy skin

disease virus (LSDV) (250). To date none of the poxvirus serpins are essential for virus

growth in tissue culture.



SERP2

Like CrmA, the myxoma virus serpin SERP2 also contains an Asp residue at the

P1 position in the RCL. SERP2 is 35% identical to CrmA, can block caspases-1 and -8

as well as granzyme B activity (424). With respect to caspase-1, SERP2 is a less

effective inhibitor (K, = 80 nM) than CrmA (K, = 4 pM). Similarly for granzyme B,

SERP2 is four times weaker (K, = 420 nM) compared to CrmA (K; = 100 nM). Deletion









of the SERP2 gene in myxoma virus leads to attenuation of disease as evidenced by

survival of infected rabbits (257). SERP2 has been reported to inhibit the influx of

inflammatory cells into the site of primary inoculation as well as prevent apoptosis of

infected lymphocytes in the rabbit model. While recombinant cowpox virus expressing

SERP2 instead of CrmA failed to block the induction of apoptosis in pig kidney cells,

SERP2 in the context of myxoma virus does inhibit apoptotic induction during infection

of rabbit kidney cells (424). The importance of the P1 residue in SERP2 is evidenced by

the observation that mutant viruses, in which the P I Asp is changed to Ala, cause an

attenuated disease (Amy MacNeill and Richard Moyer unpublished). If SERP2 were to

function simply as a caspase inhibitor in vivo, then a recombinant myxoma virus

expressing CrmA instead of SERP2 should produce similar effects as the wt myxoma

infection. On the contrary CrmA replacements of SERP2 in myxoma virus causes only

~60% mortality with a marked difference in primary lesion pathology (Amy MacNeill

and Richard Moyer unpublished). Thus SERP2 is an important virulence factor for

myxoma and deducing its natural target would provide clues to its role during myxoma

infection as well as its function as a serpin.



Cytokine Response Modifier A (CrmA)

CrmA was the first viral serpin molecule identified (316). It is an example of a

cross class inhibitor in that it has been shown to inhibit both the serine protease granzyme

B as well as members of the caspase family of cysteine proteases (201). CrmA was first

identified based on its ability to alter lesion morphology on chicken chorioallantoic

membranes (CAM). CPV produces characteristic red, hemorrhagic, non-inflammatory








pocks or lesions on 11 day old CAMs. Deletion of the CrmA gene resulted in a mutant

virus that produces white, non-hemorrhagic pocks that contained infiltrating

inflammatory cells consisting mainly of heterophils (analogous to neutrophils in

mammals) (306). Thus the gene responsible for this prevention of heterophil influx was

termed cytokine response modifier-A (CrmA). White pocks also produce much less virus

compared to wt CPV and have the ability to reduce nitroblue tetrazolium (NBT)

consistent with the presence of activated chicken heterophils. Histological analysis of the

white pocks also shows a moderate heterophilic inflammation absent in wt CPV

infections. Another study has shown that CrmA prevents the formation of (14R, 15S)

dihydroxyeicosatetraenoic acid (diHETE), an arachidonic acid metabolite during CPV

infection (305). It was further suggested that these diHETE intermediates could directly

or indirectly lead to the generation of inflammatory mediators. Whether or not this is the

case during CPV infections of CAMs has not yet been established.

Around the same time it was shown that CrmA could inhibit caspase-1 (IL-

lp convertase) in vitro (331). Caspase-1 plays a major role in the maturation ofproIL-l1

and prolL-18, both of which are proinflammatory cytokines (122, 133,413). Therefore

the role played by CrmA in blocking inflammation during CPV infections of CAMs was

thought to be through inhibition of a caspase-1 mediated release of proinflammatory IL-

1p3 and/or IL-18. IL-18 was previously described as the factor that induces synthesis of

interferon y (IFNy). Viral proteins that function to block the activity of both IL-1 3 and

IFNy provide evidence for the importance of these host responses to poxvirus infections.

In addition to controlling IL-1p3 maturation through CrmA, orthopoxviruses also encode a

soluble IL-1p receptor that binds IL-13 (381). Furthermore it is this soluble secreted









receptor, not CrmA that serves to control fever mediated by IL-1 3 in infected mice (192).

Like IL- I3, the significance of IFN in poxvirus infections can be inferred because

poxviruses control IFN both extracellularly by encoding an IFN a/P and y binding

proteins and intracellularly through proteins (vaccinia E3L and K3L genes) that interfere

with signaling events which occur after receptor binding (9, 11, 79). Deletion of either

the soluble receptors or the intracellular viral proteins E3L and K3L also leads to

attenuation of the virus (28, 456).

In addition to caspase-1, CrmA inhibits caspases-4, -5, -8, -9 and -10 at

physiological levels (118). Caspases play a central role in mediating apoptosis.

Consistent with its ability to directly inhibit a number of apical caspases, CrmA has been

shown to block the induction of apoptosis by various stimuli in different systems (117,

171,287, 332, 407,408). Caspases can also be activated by granzyme B, a serine

protease that is part of granules synthesized by CTLs and NK cells. CrmA blocks

granzyme B mediated apoptosis by directly inhibiting granzyme B (322). Although

CrmA is shown to block the activation of caspases, its role in preventing the induction of

apoptosis during CPV infection has only been demonstrated in pig kidney cells (236,

332). Furthermore, no relationship has been found between CrmA expression and virus

yield in tissue culture as both wild type CPV and CrmA deletion mutants produce similar

amounts of infectious virus (332) and CrmA is not required for viral growth in cell

culture.









SPI-2 is a CrmA Homologue

The 38 kDa CrmA homolog found in other orthopoxviruses is designated as

serine protease inhibitor-2 (SPI-2). SPI-2 from rabbitpox virus (RPV) is 92% identical to

CrmA and its expression profile is similar to CrmA in virus-infected cells (236). In vitro

studies show both proteins inhibit caspase-1 with similar efficiencies. In pig kidney cells

CrmA functions to prevent the induction of apoptosis during CPV infections. But wild

type RPV despite the presence of SPI-2 cannot block apoptosis in the swine cells. In

mixed infection experiments, CrmA was able to substitute for SPI-2 in RPVASPI-2

infected cells, but the converse was not true. SPI-2 in RPV could not prevent apoptotic

induction in CPVAcrmA infections. Thus, in spite of their high degree of homology and

their identical properties in vitro, SPI-2 and CrmA do not seem to be functionally

equivalent. Ectromelia virus (EV) SPI-2 shares 94% identity with other poxviral

homologs (425). Similar to CrmA expression of EV SPI-2 also protected cells from TNF

mediated apoptosis and inhibited the activity of caspases-1 and -8.

While most of the information about CrmA has been obtained by ectopic

expression or virus infection of tissue culture cells, its role in vivo is less well understood.

Intranasal infections of mice with CPV deleted for CrmA were attenuated and showed a

higher LD50 by at least one order of magnitude (412). Mice infected with a CrmA

deletion mutant also had decreased pulmonary pathology and reduced inflammation

compared to wild type CPV infections. Conversely, deletion of the CrmA homolog SPI-

2, in vaccinia virus has no effect on virulence in mice (192) despite the fact that vaccinia

SPI-2 functioned to inhibit the induction of apoptosis during virus infection and virus

infected cell extracts were able to block the processing of proIL-1 P. Mice infected via








the intra dermal route with vaccinia virus deleted for SPI-2, showed enhanced pathology

(417). Surprisingly the lesions produced by the SPI-2 deleted virus were significantly

larger than those produced by wild type vaccinia. Thus although SPI-2 and CrmA are

homologous proteins, some differences do exist.



RPV SPI-2 and CPV CrmA Equivalency

Previous studies have shown that LLC-PK1 pig kidney cells infected with wt

CPV maintain a typical morphological structure of the nuclei throughout the cycle of

viral infection until cell lysis occurred (332). However pig kidney cells infected with a

CPVAcrmA mutant virus showed cytopathic effects consistent with apoptotic death.

Studies with RPV infected cells showed that RPVASPI-2 mutant virus infections also

induced typical morphological characteristics associated with apoptosis (236). But more

interestingly infections with wt RPV induced apoptosis. This result was surprising due to

the fact that RPV encodes a functional SPI-2 (CrmA) protein.

Apoptotic cells manifest a number of biochemical changes that include activation

of caspase enzymes, which lead to the cleavage of nuclear proteins such as PARP and

lamin. CPVAcrmA, RPV and RPVASPI-2 infected extracts contain an activity that

cleaved PARP from its native 116 kDa form to a 85 kDa fragment characteristically seen

in cells undergoing apoptosis whereas wt CPV infected extracts failed to do so (236).

The presence of lamin A cleaving activity in the extracts mirrored the results obtained

with PARP cleavage. These biochemical assay results correlated exactly with our

observations of infected cells stained with DAPI, which stains DNA of the cell nucleus.









SPI-2 of RPV shares 92% amino acid homology with CrmA, as shown in Figure

7. However these two proteins differ in the reactive site loop at positions P5 and P6,

thereby not excluding the possibility that the two proteins may have different inhibitory

specificities with respect to activated caspases in apoptotic cells. It has been reported that

there is at least one example of a caspase that recognizes residues away from the P1

cleavage site as far as the P5 residue. Therefore it is possible that the differences

between SPI-2 and CrmA at P5 and P6 may in fact play a role during virus infection.

Published reports from our laboratory have shown that caspases are activated during RPV

but not CPV infections (236). In vitro experiments with purified SPI-2 and CrmA have

indicated that both the proteins could inhibit the activity of caspase-1 with similar

efficiencies. These results suggest that although the two proteins SPI-2 and CrmA

function similarly in vitro, they may not be functionally equivalent in vivo.

Swapping the two genes between the viruses allows us to examine any differences that

might be present between SPI-2 and CrmA. If SPI-2 and CrmA have distinct properties,

then one would expect an infection of LLC-PK 1 cells where CrmA is expressed in place

of SPI-2 in RPV to inhibit the induction of apoptosis (unlike wt RPV infected cells). And

similarly SPI-2 expressed in place of CrmA in CPV should induce apoptosis in LLC-PK1

cells (unlike wt CPV infections). However if there were an additional factor unique to

CPV that is required in addition to CrmA to inhibit the induction of apoptosis, then

blockage of apoptotic induction would be observed only during CPV infection regardless

of SPI-2 or CrmA expression. The results of this project are presented in this thesis and

are discussed.







83





Alignment of SPI-2 and CrmA

1 mdifreiassmkgenvfispasissvltilyygangstaeqlskyvekee 50
III III I IIII1111111I111111111111 IlIi II
1 mdifreiassmkgenvfisppsissvltilyygangstaeqlskyvekea 50

51 nmdkvsaqnisfksinkvygrysavfkdsflrkigdkfqtvdftdcrtid 100

51 dknk...ddisfksmnkvygrysavfkdsflrkigdnfqtvdftdcrtvd 97

101 ainkcvdiftegkinplldeqlspdtcllaisavyfkakwltpfekefts 150
III1 IIIIII l I 111 IlIllIlII II I I111 I111 II
98 ainkcvdiftegkinplldeplspdtcllaisavyfkakwlmpfekefts 147

151 dypfyvsptemvdvsmmsmygkafnhasvkesfgnfsiielpyvgdtsmm 200
III IIIIII I II111 .1111I11II 1I I I II11 II .
148 dypfyvsptemvdvsmmsmygeafnhasvkesfgnfsiielpyvgdtsmv 197

201 vilpdkidglesieqnltdtnfkkwcnsleatfidvhipkfkvtgsynlv 250
111I 111IIII 11I11III111.:: I I11111 I I 1II
198 vilpdnidglesieqnltdtnfkkwcdsmdamfidvhipkfkvtgsynlv 247

251 dtlvksgltevfgstgdysnmcnldvsvdamihktyidvneeyteaaaat 300

248 dalvklgltevfgstgdysnmcnsdvsvdamihktyidvneeyteaaaat 297

301 svlvadcastvtnefcadhpfiyvirhvdgkilfvgrycspttnc* 346
lIi I II IIII IIIIIIII11111II11I11111111I
298 calvadcastvtnefcadhpfiyvirhvdgkilfvgrycspttn*. 342
4
PI


Figure 7. Comparison of SPI-2 and CrmA peptide sequences. P 1 position is indicated
by arrowhead. Sequence alignment was performed using GCG Gap program.









In Vivo Role of CrmA During CPV Infections of CAMs

Apoptosis and inflammation are both important mechanisms by which the host

limits and clears viral infections. The results from this project will help gain a better

understanding of viral pathogenesis in terms of both virus-host interactions and the ability

of virus to modulate the host immune system during an infection.

We use the infection of CAMs of embryonated eggs as a model to study the role

of CrmA on the innate immune response including inflammation and apoptosis. The 10

day old CAM is an excellent model to study an acute inflammatory response to infection.

At this stage of embryonic development there is an absence of circulating mature B or T

cells (63,247), minimal complement protein (116) and the inflammatory response due to

CPV infection is mainly mediated by heterophils (analogous to mammalian neutrophils)

and monocytes (112). In order to understand the role played by CrmA during a CPV

infection of the CAM, we addressed the following questions (i) does CrmA prevent white

pock formation by protease inhibition, (ii) if so, is the function of CrmA solely dependent

on inhibition ofcaspases and (iii) can serpin homologs found in different poxviruses

function similarly?

The first question was addressed by mutating the P 1 Asp residue in the CrmA

RCL to alanine (D303A), which should abolish serpin function with the predicted

consequence of inducing white pocks. Such a result would also mean that an intact

Pl Asp is critical for CrmA's function as an inhibitor of inflammation. The second

question was addressed by replacing CrmA in CPV with other caspase inhibitors such as

P35 ofbaculovirus or SERP2 from myxoma virus. If the in vivo role of CrmA was to

only inhibit caspases, then P35 and SERP2 should completely replace the function of




Full Text
124
previous reports (Fig. 12) (236). However CrmA replacements of SPI-2 in RPV, looked
similar to wt CPV infections suggesting that SPI-2 and CrmA did not function in an
identical fashion within the context of RPV infections (compare Fig. 12 and 13). These
observations strengthened our hypothesis that despite their high degrees of similarity,
SPI-2 and CrmA were not functionally equivalent. In order to confirm our results seen
morphologically by DAPI stains, we also analyzed virus-infected cell extracts for caspase
induction that would demonstrate the biochemical changes seen in apoptotic cells (see
below).
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Inhibit Caspase Activation
The induction of apoptosis is characterized biochemically (most of the time) with
the activation of caspases. To assess caspase activity in virus-infected cells, extracts
were made at different times post infection. Cell extracts were assayed for the ability to
cleave Ac-DEVD-AMC, a fluorogenic peptide that is specific for terminal caspase-3-
like enzymes (Figure 14 and 15). Cleavage of Ac-DEVD-AMC is indicative of caspase
activity typically present in apoptotic cells. Extracts were made from wt and recombinant
RPV infected cells and assayed for caspase activity by incubating a portion of the extract
with the fluorogenic peptide Ac-DEVD-AMC (Fig. 14). Peptide cleavage was
determined as a function of time and expressed as rate of cleavage per second. RPV
containing functional SPI-2 gene induced caspase activity (Fig. 14). Deletion of SPI-2
from RPV induced Ac-DEVD-AMC cleaving activity after 6 hours post infection with a
maximum rate at 12 hours post infection as shown in Figure 14. We also observed that
the rate of Ac-DEVD-AMC cleavage increased with time and stabilized at late times post


222
290. Nicklin, M. J., D. E. Hughes, J. L. Barton, J. M. Ure, and G. W. Duff. 2000.
Arterial inflammation in mice lacking the interleukin 1 receptor antagonist gene.
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291. Ninomiya-Tsuji, J., K. Kishimoto, A. Hiyama, J. Inoue, Z. Cao, and K.
Matsumoto. 1999. The kinase TAK1 can activate the NIK-I kappaB as well as
the MAP kinase cascade in the IL-1 signalling pathway. Nature 398:252-256.
292. Novick, D., S. H. Kim, G. Fantuzzi, L. L. Reznikov, C. A. Dinarello, and M.
Rubinstein. 1999. Interleukin-18 binding protein: a novel modulator of the Thl
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293. O'Neill, L. A. 2000. The interleukin-1 receptor/Toll-like receptor superfamily:
signal transduction during inflammation and host defense. Sci. STKE. 2000:RE1.
294. Ogura, Y., N. Inohara, A. Benito, F. F. Chen, S. Yamaoka, and G. Nunez.
2001. Nod2, aNodl/Apaf-1 family member that is restricted to monocytes and
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296. Okamura, H., H. Tsutsi, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto,
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cytokine that induces IFN-gamma production by T cells. Nature 378:88-91.
297. Olie, R. A., F. Durrieu, S. Cornillon, G. Loughran, J. Gross, W. C.
Farnshaw, and P. Golstein. 1998. Apparent caspase independence of
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298. Olson, T. S. and K. Ley. 2002. Chemokines and chemokine receptors in
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299. Orth, K., A. M. Chinnaiyan, M. Garg, C. J. Froelich, and V. M. Dixit. 1996.
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300. Orth, K., K. O'Rourke, G. S. Salvesen, and V. M. Dixit. 1996. Molecular
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301. Ozinsky, A., D. M. Underhill, J. D. Fontenot, A. M. Hajjar, K. D. Smith, C.
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between toll-like receptors. Proc. Natl. Acad. Sci. U. S. A 97:13766-13771.


54
factor receptor (TNF-R) superfamily. Members of the DR family include Fas/CD95,
TNF-R1, DR3, DR4 and DR5 (188). The common feature of these molecules is the
presence of the cytoplasmic death domain (DD) that is essential for apoptotic signal
transduction. Receptor binding leads to oligomerization and formation of the death
inducing signaling complex (DISC) that allows the recruitment of intracellular adapter
proteins to the DISC.
Interaction between the death domains of the receptors and adapter proteins such
as Fas associated death domain protein (FADD) and TNF-R associated death domain
protein (TRADD) are responsible for the binding. The adaptor molecules also contain
the death effector domains (DED) that allow recruitment of procaspases-8 and -10 to the
DISC. The initiator caspases are autocatalyzed and serve as activators of the terminal
effector caspases that cleave the death substrates. The importance of adapter molecules
like FADD in apoptosis is evidenced in cell lines derived from FADD knockout mice that
fail to undergo apoptosis induced by CD95 ligation. However FADD'7' fibroblasts were
still sensitive to cell death induced by drugs that utilize the mitochondrial pathway (470).
The discovery of viral and cellular proteins that function to block the signal
transduction events downstream of the DISC gave rise to a new family of anti-apoptotic
proteins termed the FLICE inhibitory proteins (FLIPs). The viral FLIP found in certain
herpesviruses and poxviruses inhibit the recruitment of procaspase-8 to the DISC
underlining the importance of this pathway in pathogen clearance. Casper/cFLIP is the
cellular homolog of vFLIP and contains two DEDs and a defective caspase-like domain.
Casper can associate with the DEDs of FADD and caspase-8 thereby interfering with the
formation of a functional DISC. As expected cells from Casper knockout mice are highly


175
NBT
+ NBT
Figure 34. CPVAIL-lf3R fails to induce inflammation during CAM infections. Pock
formation by CPVAIL-ipR on the CAMs of 11 day old chicken embryos, 3 days post
infection. Membranes were untreated or stained with NBT to show the presence of
activated heterophils.


193
Furthermore, this would suggest a novel mechanism for pro-inflammatory IL-ip
induction that can be blocked by CrmA but is independent of its anti-caspase function.
Genetic space is limited for infectious agents such as poxviruses. It is therefore
not surprising that some of the poxviral serpins are bi-functional. Although the natural
target for the poxviral serpin SPI-3 of CPV is not known, studies have shown that SPI-3
functions as a serpin in vitro (420) and also controls cell fusion in a serpin independent
manner (423). Similarly CrmA could be functioning to inhibit the induction of apoptosis
by its direct interaction with caspases and could be regulating inflammation by a yet
unknown target or mechanism.
Future Studies
CrmA definitely has a role during CPV infection. Its precise function during
virus infection is not yet known and the natural target for CrmA is yet to be elucidated.
Owing to the limited availability of reagents for chickens, it will be useful to use the
mouse models of infection to answer some of these questions. A number of mouse
models of infection have been discussed that measure inflammation. Not all of these
have been tested for the effect of the CrmA gene during virus infection. As mentioned
earlier, deletion of CrmA in the intranasal model shows only mild attenuation while
deletion of SPI-2 has no effect on infection (193, 412). Most recently mice infected via
the intradermal route in the ear, show an increase in lesion pathology due to the absence
of SPI-2 (418). Studies from our lab show promise for the use of an intratracheal route
during CPV infection (Amy MacNeill and Richard Moyer unpublished). Furthermore a
number of knockout mice for the different components of the IL-1 (Table 1) and IL-18


CHAPTER 1
LITERATURE REVIEW
Introduction
Poxviruses are unique DNA viruses in that they replicate exclusively in the
cytoplasm of infected cells. Smallpox, caused by variola virus, is by far the most
infamous poxvirus and has been associated with human infections for about 2000 years
(102). The ability of poxviruses to evade the effectors of antiviral immunity is evidenced
by numerous poxvirus proteins that interact with the host immune system (271).
Studying the interactions of viral proteins would be useful in understanding the immune
response to a poxvirus infection and would also help in elucidating mechanisms of
immune evasion used by poxviruses. Before discussing the role of poxvirus proteins in
modulating the host immune response, it is necessary to review basic aspects of innate
immunity, mediators of inflammation, apoptosis and poxvirus biology.
Immune Systems
The mammalian immune system is divided into the innate and adaptive
components. Innate immunity provides defense against a wide variety of pathogens
without requiring prior exposure, involves an inflammatory response and takes place at
the local site of immune challenge (reviewed in 172, 293). Although innate immunity
was thought to be nonspecific, considerable advances in our understanding of the innate
immune system have made it clear that this response can also recognize self from non-
1


53
A\\im and facilitating the release of mitochondrial pro-apoptotic proteins into the
cytoplasm.
The release of cytochrome c leads to formation of the mitochondrial apoptosome
in conjunction with cytoplasmic Apaf-1 and procaspase-9, thus activating caspase-9.
Second mitochondrial activator of caspases (SMAC) or direct IAP binding protein with
low pi (Diablo), is a 25 kDa pro-apoptotic polypeptide released from mitochondria (87,
434). The first four amino acids of mature SMAC/Diablo, Ala-Val-Pro-Ile (AVPI) binds
baculovirus inhibitor of apoptosis repeat (BIR) 3 domain of X chromosome encoded
inhibitor of apoptosis protein (XIAP) (59). SMAC/Diablo can also bind the BIR2
domain of XIAP, a region important for XIAP mediated inactivation of caspases-3 and -
7. Thus SMAC binding to XIAP releases active caspases-3, -7 and -9 thereby promoting
cell death. The serine protease Htra2/Omi is also released from mitochondria and has
been shown to interact with XIAP and relieve inactivation of caspases (394). Apoptosis
inducing factor (AIF) and endonuclease G (EndoG) have recently been implicated in cell
death independent of caspase activation (224, 309, 393). Thus as a result of
mitochondrial dysfunction cell death occurs that is both dependent and independent of
caspase activity.
Extrinsic Activation of Caspases: Death Receptor Mediated Signaling
Apoptotic death, an essential feature of the mammalian immune system, regulates
lymphocyte maturation, receptor repertoire selection, homeostasis and cell-mediated
immune response. This pathway of programmed cell death makes use of specialized cell
surface receptors termed the death receptors (DRs) that belong to the tumor necrosis


49
cytoplasmic protein Bid, which in turn activates the mitochondrial pathway (223).
Similarly activated caspase-10 has been shown to cleave procaspases-3, -7 and -8 (103).
Apoptosis can also be triggered without engaging the DRs, but instead causing
mitochondria to release proapoptotic molecules into the cytoplasm. One of these is
cytochrome c, a protein involved in electron transport, that now accumulates in the
cytoplasm and binds the apoptotic protease activating factor -1 (Apaf-1), a cytoplasmic
protein that shares homology with the C.elegans ced-4 apoptosis regulator. Cytochrome
c stimulates the formation of the mitochondrial apoptosome consisting of cytochrome c,
Apaf-1 and caspase-9. The binding of procaspase-9 to oligomers of Apaf-1 occurs
through the CARD regions of these two proteins. Like caspase-8, caspase-9 also
undergoes autoactivation induced by proximity of high concentrations of the proenzymes.
Caspase-9 processes the effector caspases-3 and -7 directly and has been shown to be the
apical caspase activated in the mitochondrial pathway responsible for the activation of
caspases-2, -6, -8 and -10 (374). Convergence of the DR and mitochondrial pathways to
cell death occurs as a result of caspase-8 mediated cleavage of cytoplasmic protein Bid,
which facilitates the release of cytochrome c from the mitochondria. This is probably as
a result of a feedback amplification loop since caspase-3 mediates activation of caspases-
2, -6, -8 and -10 following activation by caspase-9 (374).
Apoptotic Effector Caspases-3, -6 and -7
Caspase-3 which was first discovered as a gene product with high homology to
the C.elegans CED-3 protein and which caused apoptosis when overexpressed in Sf-9
insect cells (104). Subsequent work showed caspase-3 to be necessary for the cleavage


153
culture there is little effect of CrmA on virus yield as virus produced by wt CPV and
CPVAcrmA are indistinguishable (332).
P35, SERP2 and CrmA Block Induction of Terminal Caspase Activity within
Infected Pocks
Inhibition of apoptosis by CrmA in CPV infected cells in culture has been
previously demonstrated (332). Based on the known inhibitory profiles of CrmA, P35
and SERP2 towards apical caspases (caspases -8 and -10), which are involved in
apoptosis, SERP2 and P35 might also be expected to inhibit apoptosis of infected cells in
vivo as well as in cell culture. Additional evidence that P35 and SERP2 inhibit induction
of terminal caspase activity and block apoptosis can be seen when these viruses are used
to infect primary chicken embryo fibroblasts (CEFs). While no induction of terminal
caspase activity was noted with CPV, CPVAcrmA ::P3 5 or CPVAcrmA ::SERP2
infections, CPVAcrmA: :lacZ induced terminal caspase activity during infection of CEFs
(Fig. 25). However, we have observed that in cell culture, involving pig kidney cells,
only P35 functions like CrmA and is able to block the induction of apoptosis within the
context of CPV infections (Fig. 13 and 15). SERP2 within the context of CPV infections
fails to prevent the induction of apoptosis in swine cells (Fig. 19 and 20).
In order to determine if infected pocks contain activated caspases, we have
assayed infected pock extracts for the ability to cleave Ac-DEVD-AMC, a fluorogenic
peptide (Fig. 26). Cleavage of Ac-DEVD-AMC is indicative of terminal caspase activity
and induction of apoptosis. While little caspase induction within pocks is noted in CPV
infections (similar to mock extracts), both CPVAcrmA: :lacZ and CPVCrmA D303A


94
horseradish peroxidase or goat anti-rabbit IgG conjugated to horseradish peroxidase
(Southern Biotechnology Associates, Birmingham, AL) secondary antibodies in blocking
buffer for 1 hour at room temperature. This was followed by 5 washes (10 minutes each)
in wash buffer. Immunoreactive proteins were visualized by enhanced
chemiluminiscence using the ECL kit (Amersham) according to manufacturers
instructions.
Pulse Labeling
Confluent layers of LLC-PK1 cells (~8 x 105) in 12-well dishes were infected
with virus at MOI of 10. At 2 hours post infection (early time point) or 15 hours post
infection (late time point) medium was removed, and 300 pi of labeling medium (Cys',
*
Met', ICN Biomedicals, Irvine, CA) was added to each well. Cells were incubated for 30
min at 37C to deplete Cys and Met. After this 30 pi of 35S-translabel (ICN) was added
to each well, and labeling of proteins was allowed for 30 minutes. After labeling,
medium was removed, and cells were washed 2 x with radiolabel-ffee medium. Cells
were overlayed with 350 pi of radiolabel-free medium and incubated for various time
points (chase times). At appropriate intervals, cells were scrapped in the wells and
transferred to microfuge tubes. Samples collected (cells+supematants) were boiled with
SDS loading buffer for 15 minutes. One hundred microliter of each the collected sample
was resolved on 10% SDS-PAGE and subjected to autoradiography.


110
was monitored using a Tecan SpectraFluor microplate reader to detect free amino methyl
coumarin, with an excitation at 380 nm and an emission at 460 nm. Values were
expressed as rates of fluorescence increase per second.
In Vivo Infection Assays using CAMs
Preparation of Chicken Chorioallantoic Membranes for Infections
Embryonated chicken eggs were obtained from SPAFAS, Inc (Roanoke, IL) and
incubated at 38.5C with 50% humidity for 11 days. The position of the air sac, blood
vessels, yolk sac and growing chick embryo was noted by candling the egg against a
source of light from a table lamp. The region of shell at the air sac was swabbed with
95% alcohol. The first incision (-0.5-1 mm) was made in the shell at the air sac region
with the tip of a modified file. The tip of the file was dipped in 95% alcohol and flamed
prior to making the incision. Care was taken not to puncture the air sac. A region (-1 cm
in diameter) about the middle of the egg (chosen based on the absence of yolk sac and
large blood vessels) was swabbed with Prepodyne solution (1% iodine) (WestAgro,
Kansas, MO) and air dried. A smaller region (-0.5 cm in diameter) was swabbed with
95% alcohol inside the area cleaned by Prepodyne. Swabbing with alcohol was repeated
approximately five times. The egg was positioned with the freshly swabbed region at the
apical surface. Very gently, a second incision (0.5-1 mm) was made in the shell at the
center of the swabbed region. Aseptic precautions were taken by flaming the tip of the
file dipped in 95% alcohol prior to making the incision. Care was taken not to puncture
the chorioallantoic membrane just beneath the shell and shell membrane. The air in the
air sac was gently removed using a rubber bulb placed at the incision made over the air


LIST OF FIGURES
Figure page
1 Signaling by TNF, IL-1R and TLR 11
2 Apoptosis 43
3 Poxvirus replication cycle 68
4 Serpins as physiological regulators 72
5 Serpin RCL 73
6 Inhibition of trypsin by alpha-1 antitrypsin 74
7 Comparison of SPI-2 and CrmA peptide sequences 83
8 Construction of recombinant RPV 120
9 Construction of recombinant CPV 121
10 Expression of SPI-2 and CrmA during RPV infections 122
11 Expression of CrmA and SPI-2 during CPV infections 123
12 Morphological characteristics of LLC-PK1 cells infected with RPV
derivatives 125
13 Morphological characteristics of LLC-PK1 cells infected with CPV
derivatives 126
14 Biochemical changes in LLC-PK1 cells infected by RPV derivatives 128
15 Biochemical changes in LLC-PK1 cells infected by CPV derivatives 129
16 Comparison of P35 expressed in RPV and CPV 133
17 Effect of AraC on P35 expression in recombinant RPV and CPV 136
18 Expression of SERP2 and CrmA D303A recombinant proteins 139
19 Morphological changes in LLC-PK1 cells infected with CPVAcrmA::SERP2
or CPVCrmA D303A 142
20 Biochemical changes in LLC-PK1 cells infected with CPVAcrmA::SERP2
or CPVCrmA D303A 143
21 Pock morphology on CAMs infected with recombinant viruses 146
22 Histological examination of pocks from infected CAMs 149
23 Inflammatory pocks show similar levels of oxidative burst 151
24 P35 and SERP2 restore viral yields from infected CAMs 154
25 P35, SERP2 and CrmA block terminal caspase activity within CEF cells 156
26 P35, SERP2 and CrmA block terminal caspase activity within infected pocks 157
27 Chicken IL-1 p processing 161
28 P35 and SERP2 function like CrmA to prevent Caspase-1 mediated
processing of proIL-1 P 163
29 P35 and SERP2 function like CrmA to prevent processing of chicken
proIL-18 in CAMs 165
30 Caspase-1 mediated processing of chicken proIL-18 is blocked by peptides 168
31 Chicken proIL-18 processing activity in CPVAcrmA::lacZ extracts is
inhibited by caspase specific peptides 169
IX


192
is able to function like SERP2 to inhibit apoptotic induction in rabbit cells (Peter Turner
and Richard Moyer unpublished). SERP2 was also shown to be a much weaker inhibitor
of caspase-1 and granzyme B than CrmA (424). Studies involving infections of
European rabbits reveal a role for SERP2 in myxoma virus virulence (257). When
SERP2 is replaced by CrmA in myxoma virus, the disease is markedly attenuated
although 60% of the rabbits do succumb to the infection (Amy MacNeill and Richard
Moyer unpublished). Interestingly the primary lesions of rabbits infected with
MYXASERP2::crmA look like those of the SERP2 deletion mutant. In addition there is
some reduced ability of recombinant myxoma expressing CrmA to cause spread and
secondary lesion development. Since CrmA is unable to completely restore SERP2
function in myxoma virus, it suggests that the function of SERP2 is more than just
inhibiting caspases. The natural target of SERP2 during myxoma virus infections is yet
to be elucidated.
The third possible reason for the inflammation on CAMs is that the chicken
precursor proIL-lp itself is biologically active. In the mammalian system the biological
activity of IL-1 is shared by IL-la and IL-1 P (84). A third member of the family is the
IL-1 receptor antagonist (IL-IRa), which blocks the activities of IL-la and IL-ip by
competing for the receptors (86). ProIL-ip is biologically inactive, whereas proIL-la is
fully functional as a precursor (82). In chickens, the homologs of IL-la or IL-IRa are
yet to be found (384). Since the IL-1 system in chickens is more closely related to that of
amphibians and fish than to that of mammals (475), it is conceivable that IL-la is not
present in chickens and thus the function of IL-1 would be mediated by IL-1 p alone.


113
cycles of freeze-thawing at -70 and 25C. The crude lysates were plaqued on CV-lcells
and viral titers were expressed as PFU/pock.
Terminal Caspase Activity Assay on Extracts from Infected CAMs
Individual pocks were isolated from infected membranes. Extracts of pocks were
made by grinding pocks using a microfuge pestle in lOOpl of extract buffer (10 mM
HEPES, pH 7.5; 2 mM EDTA; 0.1% CHAPS; 1 mM dithiothreitol [DTT]), freeze
thawing three times and clarifying by centrifugation at 12,000 x g for 5 minutes. The
protein concentrations in collected supernatants were determined by Bradford assay in a
microplate reader. Terminal caspase activity was determined as increase in fluorescence
with time of the substrate Ac-DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl
coumarin)] (Bachem) used at 10 pM with 25 pg of pock extract in 200 pi of caspase
buffer (100 mM HEPES, pH 7.5; 10% sucrose; 0.1% CHAPS; 10 mM DTT). Cleavage
of the substrate was monitored using a Tecan SpectraFluor microplate reader to detect
free amino methyl coumarin, with an excitation at 380 nm and an emission at 460 nm.
Values were expressed as rates of fluorescence increase per second.
Alternatively, CEF cells were grown to 80% confluency in 12 well dishes
(Costar). The cells were infected with virus at a multiplicity (MOI) of 10. After 2 hours
of absorption (9 hours for CEF cells) the inoculum was removed and fresh Medium 199
without serum was added. The cells and supernatants were harvested at different time
points and lysed in extract buffer. The extracts were processed for terminal caspase
activity using the substrate Ac-DEVD-AMC.


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pathway in mouse EL-4 cells. Biochem. Biophys. Res. Commun. 244:183-186.
201. Komiyama, T., L. T. Quan, and G. S. Salvesen. 1996. Inhibition of cysteine and
serine proteinases by the cowpox virus serpin CRMA. Adv. Exp. Med. Biol.
389:173-176.
202. Kopp, E., R. Medzhitov, J. Carothers, C. Xiao, I. Douglas, C. A. Janeway,
and S. Ghosh. 1999. ECSIT is an evolutionary conserved intermediate in the
Toll/IL-1 signal transduction pathway. Genes Dev. 13:2059-2071.
203. Kostavasili, I., A. Sahu, H. M. Friedman, R. J. Eisenberg, G. H. Cohen, and
J. D. Lambris. 1997. Mechanism of complement inactivation by glycoprotein C
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204. Kotwal, G. J., S. N. Isaacs, R. McKenzie, M. M. Frank, and B. Moss. 1990.
Inhibition of the complement cascade by the major secretory protein of vaccinia
virus. Science 250:827-830.
205. Kouroku, Y., E. Fujita, A. Jimbo, T. Kikuchi, T. Yamagata, M. Y. Momoi, E.
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Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation.
Hum. Mol. Genet. 11:1505-1515.
206. Krathwohl, M. D., R. Hromas, D. R. Brown, H. E. Broxmeyer, and K. H.
Fife. 1997. Functional characterization of the CC chemokine-like molecules
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S. A 94:9875-9880.
207. Krippner-Heidenreich, A., R. V. Talanian, R. Sekul, R. Kraft, H. Thole, H.
Ottleben, and B. Luscher. 2001. Targeting of the transcription factor Max
during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual
glutamic acid residue in position PI. Biochem. J. 358:705-715.
208. Kroemer, G., N. Zamzami, and S. A. Susin. 1997. Mitochondrial control of
apoptosis. Immunol. Today 18:44-51.
209. Kuida, K., J. A. Lippke, G. Ku, M. W. Hardinng, D. J. Livingston, M. S. S.
Su, and R. A. Flavell. 1995. Altered cytokine export and apoptosis in mice
deficient in interleukin-1 p converting enzyme. Science 267:2000-2003.
210. Kumar, M. and G. G. Carmichael. 1998. Antisense RNA: function and fate of
duplex RNA in cells of higher eukaryotes. Microbiol. Mol. Biol. Rev. 62:1415-
1434.
211. L'Heureux, G. P., S. Bourgoin, N. Jean, S. R. Mccoll, and P. H. Naccache.
1995. Diverging signal transduction pathways activated by interleukin-8 and


131
P35, when expressed in the context of CPV, was seen to migrate at 35 kDa and
appear as a diffuse immunoreactive band present only in CPVAcrmA::P35 infections as
early as 2 hours post infection (Fig. 16B). But unlike in the case of RPVASPI-2::P35
infections (Fig. 16A), P35 expressed in CPVAcrmA::P35 was stable throughout the
course of infection up to 16 hours post infection (compare Fig. 16A and 16B, lanesl-
7). In order to ascertain if the differences in P35 expression seen within the context of
RPV and CPV infections are related to apoptosis, we analyzed infected cells for
morphological and biochemical features of apoptotic induction.
RPVASPI-2::P35 Induces Apoptosis in LLC-PK1 Cells
The reason for replacing SPI-2 in RPV with P35 was because P35 is a pan-
caspase inhibitor and functions to prevent the induction of apoptosis in a number of
diverse systems (250). It was rather unexpected to note that the levels of P35 decreased
after 8 hours of recombinant RPV infection (Fig. 16A, lanes4-7). To investigate if this
observation was related to apoptosis, we tested infected cells for morphological changes
associated with apoptosis (Fig. 12, E) and infected cell extracts for caspase activity
(Fig. 14).
DAPI staining of RPVASPI-2::P35 infected cells at 16 hours post infection
revealed the presence of densely staining apoptotic nuclei similar to those seen in RPV
and RPVASPI-2 infections (Fig. 12). This result implied that in the context of RPV, P35
was unable to block the induction of apoptosis. To test if the morphological observations
of infected cells correlated with biochemical activity of caspases within infected cells, we
analyzed infected cell extracts for the ability to cleave Ac-DEVD-AMC substrate (Fig.


161
1 2 3 4 5 6
Figure 27. Chicken proIL-P processing. S labeled chicken proIL-ip proteins
synthesized in vitro were incubated with either buffer (lane 1), 1U of human caspase-1
(lane 1) or with 200 pg of extracts from confluently infected CAMs harvested 48 hours
post infection. Lanes: 1, buffer or 1U caspase-1; 2, CPV; 3, CPVAcrmA::lacZ; 4,
CPVAcrmA::P35; 5, CPVAcrmA::SERP2; 6, CPVCrmA D303A. The protein mixtures
were resolved by electrophoresis on 10% SDS-PAGE and visualized by autoradiography.
Radiolabeled peptides are: chicken proIL-ip 29 kDa.


171
Caspase-3 Activity in the Presence of CAM Extracts
In order to eliminate the possibility that the extracts might contain an endogenous
caspase-3 inhibitor, we added exogenous caspase-3 to CPVAcrmA::lacZ lysates and
assayed for the ability of the protein mixtures to cleave the caspase-3 substrate Ac-
DEVD-AMC fluorometrically. As seen in Figure 32 when 10U of recombinant human
caspase-3 was added exogenously to CPVAcrmA::lacZ extracts, there was an increased
effect on the rate of Ac-DEVD-AMC cleavage that was due to the combined activities of
either caspase-3 or extracts tested alone. From this result we find no evidence of
CPVAcrmA::lacZ infected CAM extracts to inhibit caspase-3 and therefore conclude that
the chicken proIL-18 processing ability found in the extracts seen in Figure 32 to be due
to a caspase-3-like enzyme.
However, it was interesting to note that both P35 and SERP2 replacements of
CPV induced an inflammatory response on the CAMs but yet extracts from these
infections failed to process proIL-1p or proIL-18. This observation suggest that IL-1 p or
IL-18 may not be the mediators of inflammation during CPV infection and opens up the
exciting possibility that CrmA functions to inhibit a novel target or activity.
CPVAIL-ipR Fails to Induce Inflammation on CAMs
IL-1 p is produced by a number of cell types and serves as proinflammatory
cytokine in response to cell injury (85). It has been demonstrated that the virally encoded
IL-ip receptor and not SPI-2/CrmA, is the molecule that is responsible for controlling the
actions of IL-1 p during vaccinia infections of mice (192). CPV also encodes a soluble
viral IL-1 P receptor that can bind IL-1 p and is thought to function as a decoy


195
negative bacteria (172). TLR2 recognizes bacterial lipoproteins from a number of Gram
positive bacteria (172). It is possible that CrmA interferes with the generation of ligands
that could trigger TLR mediated innate response. CPV contains homologs to A46R
(ORF VI76 in CPV) and A52R (ORF VI82 in CPV), two proteins found in vaccinia
virus that has been shown to antagonize IL-1, TLR and IL-18 signaling (172,
http:://www.poxvirus.org). If TLRs play a role in mediating inflammation during CPV
infection of the CAM, then it is possible that VI76 or VI82 gene products may also be
involved in blocking this host immune response. A mutant CPV lacking either VI76 or
V182 could be expected to induce an inflammatory reaction during CAM infection and
thereby produce white pock phenotype.
We were unable to detect caspase-1 activity under any infection condition.
However, a number of alternate proIL-ip and proIL-18 processing mechanisms have
been discussed earlier. There exists a possibility that CrmA could be involved in blocking
the activity of a non-caspase enzyme that processes precursor IL-1 p or IL-18. Neutrophil
enzyme PR-3, has recently been shown to generate IL-18 (99, 389), it is yet to be seen if
CrmA can interfere with PR-3 activity. Similarly, a number of matrix metalloproteases
(MMP) have been shown to process proIL-1 P (355). Indeed homologs of some of these
MMPs have been identified in chickens (64, 137, 328). The ability of CrmA to inhibit
avian MMP activity can be tested.
It remains a possibility that during the course of future studies novel targets for
CrmA interaction will be discovered.


17
studies underscore the importance of IL-1 as a key proinflammatory cytokine that
mediates systemic effects and rapidly activates the innate immune response.
The end result of IL-1 signaling is the expression of genes, most of which are
involved in immunity and inflammation (81). IL-1 induces expression of a number of
cell proliferation factors such as granulocyte colony stimulating factor (G-CSF),
granulocyte-monocyte colony stimulating factor (GM-CSF) and monocyte colony
stimulating factor (M-CSF). In addition IL-1 also stimulates platelet production and
increases prostaglandin E synthesis. The production of a number of cytokines involved
in generating an inflammatory response is also stimulated by IL-1 including IL-1, -2, -3, -
6, -7, -8 and TNF. IL-1 can also activate natural killer (NK) cells, T- and B-cells. IL-1 is
a potent fever-inducing agent (81). The activities of IL-1 demonstrate its role in
inducing an inflammatory response. The crucial role of IL-1 in forming part of the innate
immune response is borne out by the fact that pathogens such as poxviruses encode
molecules that interfere with IL-1 P function. The CrmA protein encoded by
cowpoxvirus is a potent inhibitor of caspase-1 thus affecting the IL-1 P maturation
process (118). In addition cowpoxvirus also encodes a (decoy) receptor that binds IL-ip
preventing interaction with cellular IL-1R (381). Both CrmA and the virus-encoded IL-
ipR molecules are the subjects of this dissertation.
Inflammatory Mediators
Tumor Necrosis Factor (TNF)
Along with IL-1, TNF is considered a primary proinflammatory cytokine.
Although mainly produced by mononuclear cells, TNF can also be produced by a variety


56
Granzyme B
Cytotoxic T cells CTLs and NK cells can selectively induce apoptosis in by
binding the death receptor CD95 on target cells or alternatively can cause apoptosis via
the cytotoxic granule granzyme B (GrB). GrB, a 35 kDa protein, is a member of the
granzyme family of lymphocyte serine proteases and is the only enzyme known to date
that shares aspartate cleaving specificity with caspases. Granzymes in general are
involved in cytolysis brought about by CTLs and NK cells. There are 5 granzymes
known to date in humans -A, -B, -H, -K and -M (184) the most abundant being GrA and
GrB (320). Granzymes are able to enter the target cells via the pore forming protein
perforin. Perforin shares structural and functional homology with the complement
membrane attack complex and gets inserted into target cell membranes where it
polymerizes to form perforin pores. Granzymes are synthesized as pre-zymogen
peptides. The pre or leader sequence targets the molecule to the secretory pathway.
Cleavage of the propeptide activates the enzymes. While purified granzymes alone have
no cytotoxic effects, their enzymatic substrates are potent cytolytic agents. GrB has been
shown to cleave procaspases-3, -6, -7, -8, -9, and -10 thus activating both initiator and
effector caspases. Cleavage of the Bcl-2 family member Bid directly by GrB activates
the mitochondrial pathway in apoptosis. In addition GrB has been shown to cleave death
substrates including PARP, DNA-PKcs, and NuMA (16, 114). Thus GrB plays an
important role in the induction of apoptosis through both the intrinsic and extrinsic
pathways.


210
156. Horai, R., S. Saijo, H. Tanioka, S. Nakae, K. Sudo, A. Okahara, T. Ikuse, M.
Asano, and Y. Iwakura. 2000. Development of chronic inflammatory
arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-
deficient mice. J. Exp. Med. 191:313-320.
157. Hoshino, K., H. Tsutsui, T. Kawai, K. Takeda, K. Nakanishi, Y. Takeda, and
S. Akira. 1999. Cutting edge: generation of IL-18 receptor-deficient mice:
evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor.
J. Immunol. 162:5041-5044.
158. Howard, J., D. E. Justus, A. V. Totmenin, S. Shchelkunov, and G. J. Kotwal.
1998. Molecular mimicry of the inflammation modulatory proteins (IMPs) of
poxviruses: evasion of the inflammatory response to preserve viral habitat.
Journal Of Leukocyte Biology 64:68-71.
159. Hsu, H., J. Huang, H. B. Shu, V. Baichwal, and D. V. Goeddel. 1996. TNF-
dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling
complex. Immunity 4:387-396.
160. Hsu, H., H. B. Shu, M. G. Pan, and D. V. Goeddel. 1996. TRADD-TRAF2 and
TRADD-FADD interactions define two distinct TNF receptor 1 signal
transduction pathways. Cell 84:299-308.
161. Hsu, H., J. Xiong, and D. V. Goeddel. 1995. The TNF receptor 1-associated
protein TRADD signals cell death and NF-kappa B activation. Cell 81:495-504.
162. Hu, F. Q., C. A. Smith, and D. J. Pickup. 1994. Cowpox virus contains two
copies of an early gene encoding a soluble secreted form of the type II TNF
receptor. Virology 204:343-356.
163. Huang, H. K., C. A. P. Joazeiro, E. Bonfoco, S. Kamada, J. D. Leverson, and
T. Hunter. 2000. The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-
protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7. J.
Biol. Chem. 275:26661-26664.
164. Huang, Y., Y. C. Park, R. L. Rich, D. Segal, D. G. Myszka, and H. Wu. 2001.
Structural basis of caspase inhibition by XIAP: differential roles of the linker
versus the BIR domain. Cell 104:781-790.
165. Huntington, J. A. and P. E. Stein. 2001. Structure and properties of ovalbumin.
J. Chromatogr. B Biomed. Sci. Appl. 756:189-198.
166. Huwiler, A. and J. Pfeilschifter. 1994. Interleukin-1 stimulates de novo
synthesis of mitogen-activated protein kinase in glomerular mesangial cells.
FEBS Lett. 350:135-138.


35
evidenced by the expression of soluble IL-1 (3 receptors encoded by poxviruses. The
vaccinia virus B15R protein based on sequence was proposed to function as an IL-1
binding protein (376). Indeed B15R was found to bind IL-1 p but not IL-1-a or IL-IRa
(8, 381). Viruses lacking B15R were found to be less virulent than wild type when
injected into weanling mice via the intracranial route. Similar experiments in mice
infected via the intranasal route with virus disrupted for B15R accelerated the illness and
mortality indicating a role for IL-1 in modulating virulence. Furthermore B15R was also
found to prevent fever caused by virus infection in mice. When the non-functional B15R
gene in Copenhagen, a virulent strain of vaccinia, was repaired, the resulting virus
infection suppressed fever and attenuated viral virulence (10). Recent evidence shows
that poxviruses can also interfere with signaling events downstream of TNFR and IL-1RI
such as NF-kB activation although observed responses seem to be specifically different
for each virus tested (295).
IL-18 Inhibitors
The central role of IL-18 in inducing IFNy that protects against bacterial and viral
infection is well documented (318). Interestingly a soluble cellular IL-18 binding protein
(IL-18BP) was identified and found to be more homologous to putative poxviral proteins
than to mammalian IL-18 receptors (292). In the cellular context IL-18BP functions as a
decoy receptor to bind IL-18 preventing its activity. Viral IL-18BPs (vIL-18BP) have
been identified in vaccinia, cowpox, ectromelia, variola and swinepox viruses (292). In
addition Molluscum contagiosum virus was found to contain three open reading frames
with homology to IL-18BP (457). Two of the Molluscum gene products MC53L and


191
an absolute requirement for the lipoxygenase pathway for replication, there is a potential
to form mediators of inflammation such as leukotrienes, which are derived from the
lipoxygenase pathway. Lipoxygenase metabolites have been shown to be both positive
and negative regulatory effectors of the immune system (323, 326, 400, 401). 12(S)-
HETE enhances tumor cell adhesion to endothelium by up regulating the expression of
integrins (401). 12(S)- and 15(S)-HETE are known to be involved indirectly in rat
carcinoma cell survival (400). Interestingly 14(R), 15(S) diHETE has been shown to
inhibit leukotriene B4 function in neutrophils (323) and natural killer cell activity (326).
It is unclear at present if any of the lipoxygenase metabolites are directly involved in the
induction of inflammation on the CAMs during CPV infection or if they serve as
intermediates in pathways that lead to the production of inflammatory mediators. While
the accumulation of diHETE during CPV and CPVAcrmA infections of CAMs was
detected as early as 1 hour post infection (305), caspase activity due to CPVAcrmA can
only be found after 8 hours of infection in tissue culture (424). Our data supports a
model of dual targets for CrmA, where the regulation of apoptosis is brought about by its
direct interaction with caspases and the regulation of inflammation is brought about by
inhibiting the formation of inflammatory mediators which may include or induce
formation of lipoxygenase metabolites. Thus using other caspase inhibitors such as P35
and SERP2, we were able to replace the anti-apoptotic function of CrmA, but were
unable to inhibit the inflammatory response on CAMs.
There is some evidence to support the dual function hypothesis. It has been
reported that SERP2 in wt myxoma virus infections, functions to prevent the induction of
apoptosis of rabbit cells (257). Similarly CrmA expressed in recombinant myxoma virus


150
Figure 22. Continued


173
receptor regulating IL-1(3 action (381). If IL-ip were to be responsible for the
inflammation on the CAMs, then one would expect a recombinant CPV deleted for the
virus encoded IL-P receptor to also induce inflammation. In order to test this hypothesis,
CPV AIL-1 PR was constructed as outlined in Figure 33.
Briefly, plasmids containing homologous regions to the vIL-ipR were
constructed, interspersed with the selectable marker Eco.gpt. Using MPA selection
insertional vIL-1 PR mutant CPV were constructed. Since we did not have reagents to
test for loss of protein expression by immunoblot analysis, our examination of the
recombinant virus was limited to analyzing PCR products for loss of the gene. As
outlined in Materials and Methods (Recombinant Virus Construction), virus plaques
resistant to MPA were selected during the first round of plaque purification. Since this
plaque pick was likely to be contaminated with wild type CPV, additional rounds of
plaque purification were necessary. The CPVAIL-ipR virus was isolated following five
such rounds of plaque purification. As shown in Figure 33, using primers that bind vIL-
1 PR, PCR products were generated from viral DNA and resolved on agarose gels. The
gene encoding vIL-1 PR (present in wt CPV) is represented by a band migrating at 1 kbp
(Fig. 33B, lane 1) present in DNA samples made from the first round of plaque
purification. However, subsequent to plaque purification, vIL-1 PR did not get amplified
from recombinant CP VAIL-1 PR virus DNA. The 1.2 kbp PCR product band present in
Figure 33B, lane 1, represents the insertion of Eco.gpt into vIL-ipR gene in CPVAIL-
1 PR. As a control for viral DNA, CrmA was amplified using specific primers (data not
shown).


39
has been found to bind complement component C3d (108). Clearly inhibiting
complement activation and lysis of infected cells allows viruses more time to replicate
and spread.
Inhibitors of Interferons
In addition to the blocking the mentioned immune mechanisms, viral products
also mediate interferon activity and will be briefly discussed here. Poxviruses encode a
number of proteins to bind both interferon type I and II. A soluble type IIFN receptor
has been identified as the vaccinia B18R open reading frame (72, 397). B18R has high
affinity for IFNa of different species, can also be found membrane bound and its absence
from vaccinia attenuated infections in the murine intranasal model. The myxoma M-T7
products are soluble type II IFN receptors that sequester IFNy (274). A unique feature of
M-T7 is that in addition to binding IFNy it also binds IL-8 and other members of the C,
CC and CXC chemokines (214). Soluble IFNy receptor homologs appear to be present in
ectromelia, cowpox and rabbitpox viruses as well (275). Indeed the vaccinia B8R protein
binds IFNy from a different species (9). Another unique molecule is the 38 kDa
glycopeptide secreted by tanapox virus (95). Unlike any other protein known to date, the
38 kDa protein binds IFNy, IL-2 and IL-5 demonstrating yet another way viral proteins
disarm multiple immune pathways with the same molecule. There also exist numerous
protein molecules encoded by different viruses that regulate the intracellular activities of
IFN that will not be discussed here. Blocking IFN and its activity is vital for virus
survival as evidenced by numerous viral strategies discovered to date.


116
recombinant human caspase-1 or 15U of recombinant human caspase-3, (kindly provided
by Nancy Thombery, Merck Research Laboratories, Rahway, N.J.) in 100 pi (final
volume) caspase buffer (100 mM HEPES, pH 7.5; 10% sucrose; 0.1% CHAPS; 10 mM
DTT) for 4 hours at 37C. The proteins were resolved on 10 % sodium dodecyl sulfate-
polyacrylamide gels (SDS-PAGE), the radioactive signal was enhanced with Amplify
(Amersham) and the proteins visualized by autoradiography.
Alternatively, confluently infected membranes (2-3 membranes for each
infection), harvested at 48 hours post infection, were homogenized in extract buffer (final
volume 800 pi), and protein concentrations were determined by Bradford assay. 200 pg
of CAM extracts were incubated with 2 pi of S-labeled proIL-1 P or proIL-18 derived
from TNT reactions in 100 pi (final volume) Caspase buffer. The protein mixtures were
incubated for 4 hours at 37C. Following incubation the proteins were subjected to SDS-
PAGE and the gels enhanced with Amplify (Amersham). Radiolabeled proteins were
visualized by autoradiography.
When using peptide inhibitors for caspases, the CAM extracts or purified human
caspase was pre-incubated with either 10 nM of Ac-DEVD-CHO [acetyl-Asp-Glu-Val-
Asp-aldehyde] (Bachem) or 100 nM of Ac-WEHD-AMC [acetyl-Trp-Glu-His-Asp-
aldehyde] (Bachem) or 200 pi of Z-VAD-FMK [benzyloxycarbonyl-Val-Ala-Asp-(OMe)
fluromethyl ketone] (Bachem) at 37C for 2 hours prior to the addition of radiolabeled
proIL-ip or proIL-18. Caspase activity was determined by the position of the cleaved,
mature cytokine products that are smaller than the radiolabeled precursor and therefore
migrate faster on SDS-PAGE.


128
Figure 14. Biochemical changes in LLC-PK1 cells infected by RPV derivatives. Pig
kidney cells were infected at MOI10 and harvested at various times post infection. Cell
extracts were made and incubated with terminal caspase substrate Ac-DEVD-AMC, a
fluorogenic peptide. Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Ac-
DEVD-AMC cleavage rates were determined at each harvested time point and are
expressed arbitrarily as fluorescence signal units (FSU) per second. Legends
corresponding to the different viruses tested are indicated.


96
RPV or CPV) at 0.05 MOI in 500 pi serum-free medium (although the inclusion of
serum in the medium has no detrimental effect). Adsorption was allowed for 2 hours at
37C. Half an hour before adsorption was complete, plasmid DNA to be used to create
recombinant virus was diluted. Approximately 5 to 10 pg of plasmid DNA was diluted
to 50 pi in sterile distilled water. In a polystyrene tube, 30 pi of Lipofectin (or
Transfectace, Life Technologies) was diluted to 50 pi. The diluted DNA and diluted
lipofectin were mixed together in the polystyrene tube and allowed to form liposomes at
room temperature for 15 minutes. When virus adsorption was complete, the inoculum
was removed, and 1 ml of fresh serum-free medium was added to each well. The DNA
complex was then slowly added dropwise to the well with infected cells. Dishes were
incubated overnight at 37C. On the following day, an additional 1 ml of medium
containing 10% serum was added to each transfected well. After incubation for a further
period of 24 hours at 37C, the cells with medium were scrapped and collected in
microfuge tubes. Cells were stored at -80C until used. Prior to making serial dilutions
and plaquing virus, the cells were sonicated for 1 min.
Selection of Recombinant Virus in the Presence of Mycophenolic Acid
Mycophenolic acid (MPA) blocks purine metabolism and inhibits the formation
of vaccinia virus plaques in a number of cell lines (96). It has previously been shown that
inhibition by MPA can be overcome by expressing the E. coli guanine phosphoribosyl
transferase gene (Eco.gpt) in recombinant vaccinia virus in the presence of xanthine and
hypoxanthine in the medium. When constructing recombinant RPV or CPV expressing
Eco.gpt, the following procedure was followed.


79
receptor, not CrmA that serves to control fever mediated by IL-ip in infected mice (192).
Like IL-ip, the significance of IFN in poxvirus infections can be inferred because
poxviruses control IFN both extracellularly by encoding an IFN a/p and y binding
proteins and intracellularly through proteins (vaccinia E3L and K3L genes) that interfere
with signaling events which occur after receptor binding (9, 11, 79). Deletion of either
the soluble receptors or the intracellular viral proteins E3L and K3L also leads to
attenuation of the virus (28, 456).
In addition to caspase-1, CrmA inhibits caspases-4, -5, -8, -9 and -10 at
physiological levels (118). Caspases play a central role in mediating apoptosis.
Consistent with its ability to directly inhibit a number of apical caspases, CrmA has been
shown to block the induction of apoptosis by various stimuli in different systems (117,
171, 287, 332, 407,408). Caspases can also be activated by granzyme B, a serine
protease that is part of granules synthesized by CTLs and NK cells. CrmA blocks
granzyme B mediated apoptosis by directly inhibiting granzyme B (322). Although
CrmA is shown to block the activation of caspases, its role in preventing the induction of
apoptosis during CPV infection has only been demonstrated in pig kidney cells (236,
332). Furthermore, no relationship has been found between CrmA expression and virus
yield in tissue culture as both wild type CPV and CrmA deletion mutants produce similar
amounts of infectious virus (332) and CrmA is not required for viral growth in cell
culture.


Caspases 44
Inflammatory Caspases-1, -4, -5, -11 and -12 46
Apoptotic Initiator Caspases-2, -8, -9, and -10 48
Apoptotic Effector Caspases-3, -6 and -7 49
Intrinsic Activation of Caspases: Mitochondrial Permeability Transition Pore 51
Extrinsic Activation of Caspases: Death Receptor Mediated Signaling 53
Granzyme B 56
Apoptosis in Avian Species 57
Viral Inhibitors of Apoptosis 58
Viral Inhibitors of Mitochondria: PT Pore Modulators and Bcl-2 Homologs 59
vLIPs: Inhibitors of DISC 60
Baculovirus Caspase Inhibitor: P35 61
Baculovirus Inhibitor of Apoptosis Protein (IAP) 62
Caspase Independent Induction of Apoptosis 64
Poxviruses 65
Poxvirus Lifecycle 67
Importance of Poxviruses 69
Serine Protease Inhibitors (Serpins) 70
Poxvirus Serpins 75
SERP2 76
Cytokine Response Modifier A (CrmA) 77
SPI-2 is a CrmA Homologue 80
RPV SPI-2 and CPV CrmA Equivalency 81
In Vivo Role of CrmA during CPV Infections of CAMs 84
2 MATERIALS AND METHODS 87
Virology 87
Cells 87
Viruses 87
Viral Stock Preparation and Quantification 88
Molecular Techniques 89
Polymerase Chain Reaction (PCR) 89
DNA Manipulation, Ligation and Transformation 91
Viral DNA Preparation 92
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 92
Immunoblot 93
Pulse Labeling 94
DNA Sequencing 94
Sequence Analysis and Database Search 95
Recombinant Virus Construction 95
Transfection of Infected Cells Using Liposomes 95
Selection of Recombinant Virus in the Presence of Mycophenolic Acid 96
Reverse Selection of Recombinant Virus using 6-Thioguanine 99
Construction of Recombinant RPV Expressing CrmA 99
Construction of Recombinant RPV Expressing P35 100
v


145
Pock Morphology of Recombinant CPV Infected CAMs
CPV produces red, hemorrhagic pocks (Fig. 2IB), while CPVAcrmA::lacZ virus,
from which CrmA was deleted, produces white pocks as expected (Fig. 21C) (306). The
white pocks produced by CPVAcrmA::lacZ also turn dark blue in the presence of NBT
indicating the presence of activated heterophil influx, consistent with previous reports
(306). Interestingly, the CrmA D303A mutant virus, in which the PI Asp was mutated,
produced white inflammatory pocks (Fig. 2 IF). This result suggests that the PI Asp
within the reactive center loop is required for CrmA to inhibit inflammation in vivo and
further that CrmA inhibits inflammation by functioning as a protease inhibitor.
Surprisingly, both P35 and SERP2 recombinant viruses produced white pocks containing
activated heterophils as suggested by positive NBT staining (Fig. 21D and 2IE).
Therefore, P35 and SERP2 fail to inhibit the inflammatory response during CPV
infections of CAMs. This was an unexpected result since both P35 and SERP2, like
CrmA have been shown to inhibit caspase-1 in vitro, which should serve to control the
processing of IL-P and IL-18, both of which are inflammatory cytokines.
All White Pocks Contain Heterophils
Histological analysis of pocks was performed in order to confirm that white pocks
which stained positive with NBT (Fig. 21C to F) were indeed inflammatory. Individual
pocks were isolated, sectioned and stained with hematoxylin and eosin. The sections
viewed microscopically are shown in Figure 22. CPV infected pocks exhibit hemorrhage
between the ectoderm and mesoderm with few heterophils (Fig. 22C and 22D).


28
The Py subunit also brings about activation of MAP kinase and then re-associates with
GDP bound a subunits. This complex activation of kinase pathways results in the
activation of transcription factors that up regulate the expression of cell adhesion
molecules, growth factors and inflammatory cytokines that facilitate the motogenic and
mitogenic effects of IL-8.
In neutrophils, the transient increase in cytosolic free calcium and activation of
PKC is responsible for granule release and superoxide production (23). The activation of
GTPases results in cytoskeletal restructuring (40, 219). CXCR1 mediated activation of
phospholypase D also leads to superoxide formation in human neutrophils (181,211).
Activation of map kinases leads to the up regulation of transcription factors (180, 414).
Binding of ligand to CXCR2 can also lead to receptor desensitization mediated by PKC
followed by receptor endocytosis and degradation, a mechanism thought to regulate IL-8
(278, 279, 461). While the activation of transcription factors AP-1 and NF-kB by TNF-a
and IL-ip up regulate IL-8, expression of IL-8 is inhibited by interferons a and p (262,
371). Since IL-1 p and TNF-a are elevated during inflammatory states and the fact that
IL-8 is synthesized by a number of cell types, highlights its important in causing local
accumulation of neutrophils in any tissue.
In summary, IL-8 functions as a chemotactic factor for leukocytes increasing
access of immune cells such as neutrophils to sites of inflammation. IL-8 enhances
adhesion molecule binding (P2 integrin) to circulating leukocytes thereby facilitating
migration and extravasation of the immune cells across blood vessel walls into inflamed
tissue. IL-8 activates neutrophils to undergo respiratory burst and degrannulation. In


13
important in interpreting the results of experiments presented in this thesis. ProIL-1p can
be released from cells by an unknown mechanism despite the absence of any secretory
signal sequences in the molecule (33). In addition, cell lysis can also cause the release of
cytoplasmic proIL-1 P during tissue damage (142). A number of proteases can be found
at sites of inflammation such as in synovial fluid, sites of neutrophil and macrophage
infiltration and at sites of tissue damage. Chymotrypsin and Stapylococcus aureus
protease are able to process the 31 kDa proIL-1P to biologically active fragments ranging
from 17 to 19 kDa whereas the Trypsin cleavage product is inactive (38). Cathepsin G,
collagenase and elastase can also process proIL-1 p to bioactive cytokine (143). In
addition, the importance of such mechanisms in vivo was evidenced by similar processing
capabilities seen in synovial and bronchoalveolar lavage fluids from patients with
inflammatory polyarthritis and sarcoidosis (143). The CTL molecule granzyme A, a
serine protease, can also generate biologically active IL-ip, although this cleavage occurs
at Argl20, four residues downstream of the Aspl 16 site used by caspase-1 (169). The
serine protease chymase, derived from dermal mast cells, also converts proIL-ip to an
active species, suggesting a role for the initiation of inflammation in the skin (261).
Matrix metalloproteinases (MMPs) have also been implicated in the processing of proIL-
ip (355). Stromelysin (MMP-3) and gelatinase-A (MMP-2 and -B (MMP-9) can
generate biologically active forms of IL-ip. Extended incubation of the mature product
with these enzymes caused degradation. In addition, none of these MMPs were able to
process proIL-la. Proteinase-3, a proteolytic enzyme from activated neutrophils, has
also been shown to produce IL-1 p (71). The balance of accessible proteases at sites of
local inflammation may regulate the availability of active IL-1 p and thus modulate acute


6
oligoadenylate synthase (OAS) which synthesizes 2-5-oligoadenylate. The
oligonucleotide activates RNAaseL that cleaves viral and cellular RNA (210) thus
blocking viral replication and inducing apoptosis. Other intracellular PAMPs include
Nodi, a cellular protein that binds to and regulates procaspase-9, an enzyme involved in
apoptotic induction (167). Nod2, a protein expressed in monocytes, activates nuclear
factor kappaB (NF-kB), a transcription factor associated with inflammation (294). The
complete range of ligands that interact with Nod proteins is currently unknown.
Extracellular PRRs include mannan-binding lectin (MBL) that binds mannose
residues that are abundant on microbial surfaces (111). Serine proteases MASP1 and
MASP2 are activated by MBL and cleave C2 and C4 proteins, thereby initiating the
complement cascade. C-reactive protein (CRP) and serum amyloid protein (SAP)
function as opsonins by binding to bacterial phosphorylcholine (361). The macrophage
mannose receptor (MMR) facilitates the phagocytosis of bacterial and fungal pathogens
(111). Macrophage scavenger receptor (MSR) binds LPS and protects against endotoxic
shock (409). Other examples of extracellular PRRs are the Toll/Interleukin-1 family of
receptors.
Toll and Interleukin-1 Receptors
Toll and Toll-Like Receptors (TLR)
The Toll/IL-IR family of receptors are conserved from flies to mammals and play
a central role in the induction of cellular innate immune responses. Drosophila Toll
ligand has been identified as Spatzle (221). The Drosophila Toll gene product was
originally found to be required for dorso-ventral polarity (139). Toll protein was later


156
hr p.i.
Figure 25. P35, SERP2 and CrmA block terminal caspase activity within CEF cells.
CEF cells were infected at MOI10 and harvested at various times post infection. Cell
extracts were made and incubated with terminal caspase substrate Ac-DEVD-AMC, a
fluorogenic peptide. Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Ac-
DEVD-AMC cleavage rates were determined at each harvested time point and are
expressed arbitrarily as fluorescence signal units (FSU) per second. Legends
corresponding to the different viruses tested are indicated.


5
Antigen present in the draining lymph nodes activates the adaptive arm of
immunity, and thus during the later stages of an inflammatory response lymphocytes are
involved. There is a delay of 4 to 7 days from the time of injury before the effects of an
adaptive response are seen. This period is sufficient for pathogens such as poxviruses to
replicate and cause systemic spread. Therefore, in such cases the innate immune
response is crucial in controlling infections during the period before an adaptive response
is seen.
Innate Immune Recognition
The mammalian innate immune response has two steps in recognizing invading
pathogens. The first involves recognition of pathogen associated molecular patterns
(PAMPs), and the second step is based on recognition of molecular markers specific for
self (172). PAMPs are conserved products of microbial metabolism that are distributed
and common among a number of pathogens. One example of a PAMP is
lipopolysaccharide (LPS) common to all gram-negative bacteria. The molecules that
recognize PAMPs are termed pattern recognition receptors (PRRs). PRRs include a
number of molecules that can be cytosolic, membrane bound and secreted in eukaryotic
cells. PRRs function to opsonize antigens, activate complement, initiate proinflammatory
and apoptotic cascades. A common PAMP associated with virus replication is the
formation of double stranded RNA that can bind and thereby activate protein kinase R
(PKR) (69). PKR phosphorylates eukaryotic initiation factor eIF-2a which serves to
block viral and cellular protein synthesis, inducing interferon responses and causes
apoptosis of infected cells (450). Double stranded RNA also activates 2-5-


221
stimulates its degradation in non-hematopoietic cells. J. Biol. Chem. 270:10439-
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279. Mueller, S. G., J. R. White, W. P. Schraw, V. Lam, and A. Richmond. 1997.
Ligand-induced desensitization of the human CXC chemokine receptor-2 is
modulated by multiple serine residues in the carboxyl-terminal domain of the
receptor. J. Biol. Chem. 272:8207-8214.
280. Murphy, P. M. and H. L. Tiffany. 1991. Cloning of complementary DNA
encoding a functional human interleukin-8 receptor. Science 253:1280-1283.
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immune recognition. J. Cell Biol. 155:705-710.
282. Muzio, M., J. Ni, P. Feng, and V. M. Dixit. 1997. IRAK (Pelle) family member
IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278:1612-
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287. Nava, V. E., A. Rosen, M. A. Veliuona, R. J. Clem, B. Levine, and J. M.
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97
To generate recombinant poxvirus expressing Eco.gpt, plasmids containing the
Eco.gpt gene cloned within the appropriate flanking regions where homologous
recombination is expected to occur were constructed. Cells infected with virus (either
RPV/CPV) were transformed with the Eco.gpt containing plasmid as described earlier.
Following transformation of cells, the virus mixture was serially diluted and inoculated
on CV-1 cells grown in 6-well dishes. Mycophenolic acid (25 pg/ml), xanthine (250
pg/ml) and hypoxanthine (15 pg/ml) were incorporated into the agar-medium just prior to
the addition of the overlay following the viral adsorption period. After 3 to 4 days of
growth, the cells were stained with neutral red, and plaques were visualized. One plaque
for each transformation reaction was picked with the help of a 500 pi micropipette and
re-suspended in 500 pi of PBS. This was termed the first plaque pick. The viral plaque
pick was sonicated, serially diluted and plaqued again on CV-1 cells and selected against
MPA. Following this first round of plaque purification, viral plaques were again picked
(second pick) and purified by another round of plaque purification in the presence of
MPA. Typically 5 such rounds of plaque purification were performed to produce pure
recombinant virus (RPV/CPV) expressing Eco.gpt.
When producing recombinant virus by transient dominant selection (TDS) as
described by Falkner and Moss (97), the Eco.gpt gene is cloned outside the flanking
regions that were used for homologous recombination. During homologous
recombination, the entire plasmid including the Eco.gpt gene is incorporated into the viral
genome under MPA selection and is maintained as an intermediate. Removal of MPA
selective pressure allows for concatemer resolution within the intermediate virus and the


21
In summary both TNF-a and -P are able to bind TNFR and signal via DD
containing proteins initiating a cascade of events leading to the induction of apoptotic cell
death whereas events leading to IkB phosphorylation results in the translocation of NF-
kB to the nucleus where it can induce transcription of genes involved in cell survival and
the proinflammatory response. In addition TNF-a also causes neutrophil and
macrophage activation and induction to synthesize nitric oxide (NO). TNF-a also
induces the synthesis of acute phase proteins such as C-reactive protein that opsonize
microbes and activate the complement pathway. Mannan-binding lectin (MBL), a PRR
that opsonizes microbes and activates complement is up regulated by TNF-a. Like IL-
1 p, TNF-a is also a fever-inducing agent. The important role of TNF, in the innate
immune system and specifically in controlling viral infections, is evidenced by pathogens
such as poxviruses encoding molecules that interfere with TNF function. Cowpoxvirus
encodes five different TNF receptor family homologs that will be discussed in more
detail later.
Interleukin-18 (IL-18)
IL-18 was originally discovered as the factor that induced interferon gamma
(IFNy) thus being critical for the control of intracellular pathogens (296). IL-18 is a
potent inducer of CTL and NK cell activity and promotes the differentiation of T cells to
the ThI phenotype (396). While the induction of IL-18 is not entirely clear, it is seen to
be involved in an immune prophylactic response to a number of bacterial and viral
infections (35, 318). Like proIL-1 p, proIL-18 does not contain a signal peptide sequence,
is intracellular and is processed by caspase-1 (122, 133). The precursor is synthesized as


127
infection (12 to 18 hours). It is apparent that SPI-2 in RPV infections is incapable of
completely blocking caspase activation. These results show that SPI-2 does function to
reduce caspase induction although SPI-2 is unable to completely block caspase induction.
More interestingly, in extracts from RPVASPI-2::crmA infections, (CrmA replacement of
SPI-2 in RPV) induction of caspase activity was completely blocked (Fig. 14). These
results suggest that CrmA functions more efficiently than SPI-2 in the context of RPV
and that unlike SPI-2, CrmA was able to completely block the activation of terminal
caspases during RPV infections of LLC-PK1 cells.
Wild type CPV infections of LLC-PK1 cells do not induce caspase activity
(Fig. 15). However CPVAcrmA::lacZ infections (like RPVASPI-2::lacZ and to a lesser
extent RPV), induce caspases as early as 8 hours post infection and reach maximum rates
at 12 hours post infection similar to those seen with RPVASPI-2. SPI-2 replacements of
CrmA in CPV (CPVAcrmA::SPI-2) function within the context of CPV to completely
block caspase induction just like wt CPV infections (Fig. 15). Therefore in CPV
infections of pig kidney cells, SPI-2 and CrmA function in a similar fashion with the
dissimilarities between the two proteins only apparent when expressed from within RPV.
SPI-2 and CrmA are similar with respect to kinetics of expression and inhibitory
profiles based on in vitro studies (236). Our results validate previous observations seen
for CrmA and SPI-2 in genetic complementation experiments (236). Thus, despite the
similarities observed between SPI-2 and CrmA in vitro, our results imply that RPV SPI-2
and CPV CrmA are not completely functionally equivalent in vivo during infections of
swine cells.


215
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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ROLE OF CrmA IN REGULATING INFLAMMATION AND APOPTOSIS DURING
COWPOXVIRUS INFECTION
By
Rajkumar Nathaniel
May 2003
Chair: Richard W. Moyer
Department: Molecular Genetics and Microbiology
Poxviruses are large DNA viruses that replicate exclusively in the cytoplasm of
the infected host cell. The importance of the host inflammatory response in controlling a
poxvirus infection is supported by the fact that approximately one third of the poxvirus
genome is directed at deflecting the early host response to virus infection. Viral serine
protease inhibitors (serpins) are proteins encoded by poxviruses that interfere with the
host immune response. Cowpox virus (CPV) encodes the serpin CrmA which has been
shown to inhibit inflammation and apoptosis during virus infection. Wild type cowpox
virus forms red, hemorrhagic, non-inflammatory pocks on the chorioallantoic membranes
(CAMs) of embryonated chicken eggs whereas CPV deleted for CrmA forms white,
inflammatory pocks accompanied by lowered yield of virus. In vitro studies have shown
CrmA to be a potent inhibitor of caspase-1, as well as caspases-8 and -10. Caspase-1 is
an enzyme responsible for the activation of the pro-inflammatory cytokines IL-1 p and IL-
18 from their inactive precursors. A CrmA-mediated block in the activation of these pro
xy


23
showing that soluble IL-18Ra only in combination with sIL-18R(3 is able to block IL-18
induction of IFNy (336). Thus the regulation of IL-18 by soluble forms of the receptors
is similar to that seen in IL-1.
The effects of receptor binding and signal transduction are similar for both IL-18
and IL-1. IL-18 signaling complex (comprising of IL-18 bound to IL-18Ra and IL-
18RP) recruits IRAK via the adapter protein MyD88 (340, 411). MyD88'/_ cells do not
respond to IL-18 (1). Upon activation, IRAK dissociates from the receptor complex and
is responsible for the activation of NIK mediated by TRAF-6 (200, 411). The signalsome
containing IkB kinases (IKK) is activated by NIK directly or alternatively by TRAF-6
activation of MEKK1 and ECSIT (42, 202, 220). The complex interaction between a
number of protein kinases culminates in the activation and translocation of transcription
factors NF-kB and AP-1 (25, 248, 340). The steps leading to the activation of NF-kB
and AP-1 seem to be identical both in IL-1 p and IL-18. IL-18 is also involved in
activating the MAPK pathway. IL-18 induced the kinase activities of MAPK and LCK in
ThI cells (419). MAPK is involvement in cell growth could possibly explain the effect
of IL-18 in activating NK and T cells (415). Apart from inducing IFNy production from
T, B and NK cells, IL-18 acts in synergy with IL-12 to induce NK cell cytotoxicity and
ThI cell differentiation (35). Signaling pathways used by IL-18 and IL-12 are quite
different. While IL-18 activates NF-kB, IL-12 activates the signal transducer and
activator of transcription (STAT) 4 (25). The lack of IFNy production by IL-18 observed
in Th2 cells may be due to the fact that TH2 cells do not express IL-18R (466). In
addition to IFNy, IL-18 is a potent inducer of the chemokines MIP-la, MCP-1, IL-8, as
well as IL-ip and TNF-a production in different cell types (321). The ability of IL-18 to


211
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32
IL-18
The chicken IL-18 gene was discovered as a result of BLAST search analysis of a
bursal expression sequence tag (EST) database (354). Comparison of the primary
sequence between the chicken and mammalian homologs revealed that the chicken
precursor contains the conserved Asp36 that is required for processing by caspase-1 (7).
A putative peptide representing residues 30 to 198 expressed with an N-terminal
histadine tag was found to induce IFNy synthesis in primary chicken spleen cells. The
IL-18 receptor and accessory signaling protein as well as the antagonist IL-18 binding
protein are yet to be identified in birds.
CXC chemokines
The chicken chemotactic and angiogenic factor (cCAF) is a 9 kDa CXC
chemokine that was originally cloned from Rous sarcoma virus transformed fibroblasts
(29, 388). Recent studies have suggested it to be a homolog of IL-8 (183). Similar to IL-
8, the chicken cytokine can also be post translationally cleaved (by plasmin) to smaller
fragments (243). Experimental evidence points to the C-terminus as being angiogenic
(243). K60 is another chicken CXC chemokine that shares 50% homology to mammalian
IL-8 (367). K60 is secreted from macrophages stimulated with LPS, IL-ip and IFNy
(367). Interestingly both cCAF and K60 are the only chicken chemokines to date that
contain the conserved ELR motif present in all mammalian chemokines that attract
neutrophils (22). A homolog of mammalian CXCRI was cloned from a chicken genomic
library (226). The chicken receptor was found to be 67% homologous to human CXCRI.
The identification of mammalian homologs of IL-8 and related CXC chemokines in birds
demonstrate the similarities between the chemotatic systems.


1 2 3 4 5 6
Figure 20. Biochemical changes in LLC-PK1 cells infected with
CPVAcrmA::SERP2 or CPVCrmA D303A. Pig kidney cells were infected at MOI 10
and harvested at 12 hours post infection. Cell extracts were incubated with terminal
caspase substrate Ac-DEVD-AMC, a fluorogenic peptide. Cleavage of Ac-DEVD-
AMC is indicative of caspase activity. Ac-DEVD-AMC cleavage rates were determined
and are expressed arbitrarily as fluorescence signal units per second. Bar numbers: 1,
Mock; 2, CPV; 3, CPVAcrmA::lacZ; 4, CPVAcrmA::P35; 5, CPVAcrmA::SERP2; 6,
CPVCrmA D303A.


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234
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antagonizing IAP proteins. Cell 102:43-53.


19
and ultimately death by multiple organ failure due to increased vascular permeability
resulting in loss of blood volume and plasma clotting proteins.
The TNF receptor, TNF-RI, has a molecular weight of 55 kDa and acts as the
main receptor type expressed by most cells (24). TNF-RI is thought to mediate most of
the proinflammatory effects of TNF-a (315). TNF receptor mediated signaling is
depicted in Figure 1. The intracellular domain of TNF-RI contains a death domain (DD)
that is required for apoptotic signaling of cell death (402). TNF-RI is always associated
with silencer of death domains (SODD), a cellular protein that functions to prevent the
inadvertent self-association of TNF-RI death domains (173). Following TNF binding to
TNF-RI, the receptor trimerizes leading to the disassociation of SODD. The adapter
protein TNF-R associated death domain (TRADD) interacts with TNF-RI via the DDs
(159). The Fas associated death domain (FADD) adapter protein that interacts with Fas
during apoptotic signaling does not directly bind TNF-RI, but can be recruited by
TRADD to the TNF-R signaling complex. (160). The association of FADD leads to the
recruitment of procaspase-8 followed by its activation and the initiation of the caspase
cascade.
The N-terminal domain of TRADD has been found to interact with the C-terminal
domain of TNF associated factor (TRAF) 2 (160). TRAF-2 plays a role in the activation
of NF-kB and Jun N-terminal kinase (JNK) by both receptors but is not involved in
apoptosis (161, 464). RIP, a cytoplasmic serine/threonine protein kinase, strongly
interacts with TRADD in the receptor-signaling complex and activates NF-kB and JNK
pathways (159, 232). However RIP disruption (RIP'/_) affects only NF-kB but not JNK


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58
Inhibitor of T-cell apoptosis (ITA) protein contains BIR domains as well as the zinc
finger motif characteristic of IAPs (176).
To date four avian caspases have been cloned and characterized. Chicken
caspases-1, -2, -3 and -6 (174, 175, 178). There are no reports to date of purified chicken
caspases showing biochemical activity. Chicken caspase-1 while being 44% identical to
human caspase-1 has two cysteine residues (QCCRG) in the active site pentapeptide
compared to its human homolog (QACRG) that has only one. The chicken homolog of
caspase-2 is 70% identical to its human counterpart and contains the conserved QACRG
catalytic site sequence. Chicken caspase-3 and -6-like activity was noted in granulse
cells treated with the apoptotic inducer okadaic acid. Both chicken caspase-3 and -6 are
>65% identical to the human enzymes with complete conservation in the active site
region. Caspase-6 gene disruptions in a chicken cell line had no effect on the induction
of apoptosis (347). Chicken caspase-6-like its mammalian counterpart was seen to
cleave lamins A and C. Available reports of apoptosis in the avian system suggest
conservation of both DR and mitochondrial pathways. The identification of novel
molecules in the future will highlight differences and help gain a better understanding of
this process in birds.
Viral Inhibitors of Apoptosis
Viruses are obligate intracellular parasites and therefore induction of an apoptotic
reaction to infection is a highly effective host innate response that controls and limits the
spread of the invading pathogen. Viruses, in order to survive have developed multiple
ways to either neutralize or delay the cell death machinery thereby maximizing the


Construction of Recombinant CPV Expressing SPI-2 102
Construction of Recombinant CPV Expressing P35 103
Construction of Recombinant CPV Expressing SERP2 104
Site Directed Mutagenesis of CrmA Gene 104
Construction of CPVCrmA D303A 105
Construction of CPVAIL-ipR Virus 105
Apoptosis Assays 106
DAPI Staining of Infected Cells 106
Preparation of Infected Cell Extracts for Caspase Activity Assay 109
Ac-DEVD-AMC Cleavage Assay for Caspase Activity 109
In Vivo Infection Assays using CAMs 110
Preparation of Chicken Chorioallantoic Membrane for Infections 110
Infecting and Harvesting CAMs Ill
Measurement of Reactive Oxygen Intermediates Ill
MTT Reduction Assay 112
Virus Infectivity Assay 112
Terminal Caspase Activity Assay on Extracts from Infected CAMs 113
Construction of Plasmids Containing Chicken ProIL-ip or ProIL-18 114
Plasmid Containing Mouse ProIL-1 p 115
Quick Coupled Transcription/Translation System 115
In Vitro Cleavage Assay for Processing ProIL-ip and ProIL-18 115
3 RESULTS 117
Equivalency of SPI-2 and CrmA 117
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Recombinant Virus Construction 118
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Prevent Apoptosis in Pig Cells 119
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Inhibit Caspase Activation 124
Recombinant RPV and CPV Viruses Expressing P35 130
RPVASPI-2::P35 Induces Apoptosis in LLC-PK1 Cells 131
CPVAcrmA: :P35 Blocks Apoptotic Induction in LLC-PK1 Cells 132
Viral Protein Expression: Effect of AraC 134
Recombinant CPV Viruses Expressing SERP2 or CrmA D303A 137
Infections of LLC-PK1 Cells with CPV Recombinants: DAPI Stained Cells 138
Infections of LLC-PK1 Cells with CPV Recombinants: Ac-DEVD-AMC
Cleavage Activity 141
Recombinant CPV Infections of CAMs 144
Pock Morphology of Recombinant CPV Infected CAMs 145
All White Pocks Contain Heterophils 145
Inflammatory Pocks Show High Levels of NADPH Oxidase 148
P35 and Serp2 Restore Viral Yields from Infected CAMs 152
P35, Serp2 and CrmA Block Induction of Terminal Caspase Activity within
Infected Pocks 153
Effect of Caspase Inhibitors on Protease Activity Present in
Infected CAMs 155
Chicken ProIL-1 p Processing Activity in CAMs 159
vi


226
336. Reznikov, L. L., S. H. Kim, L. Zhou, P. Bufler, I. Goncharov, M. Tsang, and
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O. Fearnhead, and C. S. Duckett. 2001. Molecular cloning of ILP-2, a novel
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4301.
338. Riedl, S. J., M. Renatus, R. Schwarzenbacher, Q. Zhou, C. H. Sun, S. W.
Fesik, R. C. Liddington, and G. S. Salvesen. 2001. Structural basis for the
inhibition of caspase-3 by XIAP. Cell 104:791-800.
339. Rieux-Laucat, F., S. Blachere, S. Danielan, J. P. De Villartay, M. Oleastro, E.
Solary, B. Bader-Meunier, P. Arkwright, C. Pondare, F. Bernaudin, H.
Chapel, S. Nielsen, M. Berrah, A. Fischer, and F. Le Deist. 1999.
Lymphoproliferative syndrome with autoimmunity: A possible genetic basis for
dominant expression of the clinical manifestations. Blood 94:2575-2582.
340. Robinson, D., K. Shibuya, A. Mui, F. Zonin, E. Murphy, T. Sana, S. B.
Hartley, S. Menon, R. Kastelein, F. Bazan, and A. O'Garra. 1997. IGIF does
not drive Thl development but synergizes with IL-12 for interferon-gamma
production and activates IRAK and NFkappaB. Immunity 7:571-581.
341. Rosengard, A. M., Y. Liu, Z. Nie, and R. Jimenez. 2002. From the Cover:
Variola virus immune evasion design: Expression of a highly efficient inhibitor of
human complement. Proc. Natl. Acad. Sci. U. S. A 99:8808-8813.
342. Rosenkilde, M. M., T. N. Kledal, H. Brauner-Osborne, and T. W. Schwartz.
1999. Agonists and inverse agonists for the herpesvirus 8-encoded constitutively
active seven-transmembrane oncogene product, ORF-74. J. Biol. Chem. 274:956-
961.
343. Rothe, M., M. G. Pan, W. J. Henzel, T. M. Ayres, and D. V. Goeddel. 1995.
The TNFR2-TRAF signaling complex contains two novel proteins related to
baculoviral inhibitor of apoptosis proteins. Cell 83:1243-1252.
344. Rothe, M., V. Sarma, V. M. Dixit, and D. V. Goeddel. 1995. TRAF2-mediated
activation of NF-kappa B by TNF receptor 2 and CD40. Science 269:1424-1427.
345. Rothe, M., S. C. Wong, W. J. Henzel, and D. V. Goeddel. 1994. A novel family
of putative signal transducers associated with the cytoplasmic domain of the 75
kDa tumor necrosis factor receptor. Cell 78:681-692.


66
Table 3. Taxonomy of poxviruses
Genera
Members
Orthopoxvirus
Cowpox, monkeypox, buffalopox,
vaccinia, Variola, rabbitpox,
camelpox, ectromelia, raccoonpox,
taterapox, volepox
Parapoxvirus
Pseudocowpox, parapoxvirus of red
deer, bovine papular stomatitis virus,
orf, paravaccinia virus
Vertebrate
poxviruses
Avipoxvirus
Canarypox, fowlpox, j uncopox,
mynahpox, pigeonpox, quailpox,
sparrowpox, starlingpox, turkeypox
Capripoxvirus
Goatpox, sheeppox, lumpy skin
disease virus
Lepori poxvirus
Myxoma virus, rabbit fibroma virus,
hare fibroma virus, squirrel fibroma
virus
Suipoxvirus
Swinepox virus
Molluscipoxvirus
Molluscum contagiosum virus
Yatapoxvirus
tanapox virus, Yaba monkey tumor
virus
Entomopoxvirus A
Anmala cuprea, Aphodius
tasmaniae, Demodema boranensis,
Dermolepida albohirtum, Figulus
subleavis, Melolontha melolontha
Insect
Poxviruses
Entomopoxvirus B
Amsacta moorei, Acrobasis zelleri,
Arphia conspersa, Choristoneura
biennis, Choristoneura conflicto,
Melanoplus sanguinipes, Oedaleus
senigalensis, Schistocera gregaria
Entomopoxvirus C
Aedes aegypti, Camptochironomus
tentans, Chironimus luridus,
Chironomus plumosus, Chironomus
attenuatus, Goeldichironomus
haloprasimus
Unclassified
poxviruses
California harbor sealpox, cotia
virus, dolphinpox, embu virus, grey
kangaroopox, marmosetpox,
Molluscum-like poxvirus, Nile
crocodilepox, mule deerpox virus
http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm


45
absolute, the two residues (P2 and P3) towards the amino side of PI are not absolute but
are important in having an impact on substrate cleavage. The P4 residue on the other
hand appears to be important in determining substrate specificity. Caspases are activated
in a sequential manner beginning with those that initiate the cascade and ending in the
activation of effector caspases that result in the cleavage of the so-called death substrates.
By nature of their cleavage specificity, most caspases are capable of auto processing.
The length and presence of certain motifs on caspase prodomains are related to
their function. Caspases with large prodomains are typically involved in the initiation of
the apoptotic cascade. The death effector domain (DED) found in caspases -8 and -10
interacts with the DED of signaling adapter proteins that are associated with death
receptors involved in extrinsic signaling. Another motif, the caspase recruitment domain
(CARD) found in caspases -1,-2, -4 and -9 functions in promoting interactions between
one another and with adapter molecules. Short prodomain containing caspases are
typically effector caspases and are usually activated by initiator caspases.
Based on substrate specificities, caspases have been divided into three major
groups (119). Group I caspases are called inflammatory caspases since they function to
activate proinflammatory cytokines and include caspases-1, -4, -5, -11 and -12.
Caspases belonging to Group III function as initiator caspases and include caspases -2, -
8, -9, and -10. The initiator caspases cleave and activate Group II effector caspases that
include caspases-3, -6 and -7. Based on optimal substrate sequence specificity, caspase-2
has been placed in group II and similarly caspase-6 has been placed in group III (119).
The dual role of caspases in mediating apoptosis and inflammation indicates that these


ROLE OF CrmA IN REGULATING INFLAMMATION AND APOPTOSIS DURING
COWPOX VIRUS INFECTION
By
RAJKUMAR NATHANIEL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003

ACKNOWLEDGMENTS
A number of people have contributed to the completion of this work. I would like
to thank my mentor Richard Moyer for allowing me to start this project and also for
helping me complete it. Dick has been extremely patient and forgiving with me these
past 6 and some years. He continuously made efforts to train me as a scientist. Dick
helped me appreciate poxviruses. The members of my committee Lung-Ji Chang, Paul
Gulig, and Michael Clare-Salzler have also been instrumental in guiding me through
these years. They have sat patiently through many more committee meetings than they
would have wanted to. I would also like to thank Dr. Richard Condit and Dr. Sue Moyer
for valuable input and suggestions during joint lab-meetings.
The members of the Moyer Lab need special mention. They have been good
friends and work colleagues. Mike Duke taught me the difference between Republicans
and Democrats. Mike and I have had many philosophical discussions about the world we
live in and I will miss those interactions. Alison Bawden, Traci Ness and Kristin Moon of
the so-called Estrogen Sanctuary made sure I wasnt naive by the time they left the lab.
Ben Luttge and Lauren Brum have been wonderful work mates and strongly encouraged
my graduation process. Peter Turner has been generous with his time and was always
there when I needed technical advice. I am grateful to Marie Becker, for reading my
manuscripts and for her mothering us in the lab. Qianjun Li has been a good friend, work
neighbor and advisor.
11

I am grateful to Joyce Conners for taking care of all the necessary paperwork. I
would also like to thank the administrative staff of the Department of Molecular Genetics
and Microbiology for their timely help.
I owe a lot to friends at the Graduate Evangelical Fellowship, who have seen me
through my graduate career. I would specially like to mention Chip Appel, whose
constant encouragement and friendship was invaluable at times. Brian and Shigeko
Jackson, Diomy Zamora, Kwabena Amphosa-Managera, Shelly Merves and Ricardo
Gomez have specially seen me through the final stages of completing this project. Their
friendship has been invaluable. My family, have patiently waited for me to finish this
degree and have been a positive influence through all these years. Finally, I would like to
express my heartfelt gratitude to my wife, Leena, for her love, patience and endurance
despite all the strain we have gone through my graduate career. Leena has truly been my
helpmate. I firmly believe in the notion that it takes a village to raise an individual.
There are many others, who have, in little ways influenced my life and shaped the way I
think. Like little drops of water that make a flowing river, these individuals have touched
my life in numerous ways.
m

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ii
LIST OF TABLES viii
LIST OF FIGURES ix
ABBREVIATIONS xi
ABSTRACT xv
CHAPTER
1 LITERATURE REVIEW 1
Introduction 1
Immune Systems 1
Innate Immunity and Inflammation 3
Innate Immune Recognition 5
Toll and Interleukin-1 Receptors 6
Toll and Toll Like Receptors (TLR) 6
Interleukin-1 (IL-1) 8
Inflammatory Mediators 17
Tumor Necrosis Factor (TNF) 17
Interleukin-18 (IL-18) 21
Interleukin-8 (IL-8) 25
Avian Cytokines 29
TNF-a 29
IL-1 30
IL-18 32
CXC Chemokines 32
Viral Inhibitors of Inflammation 33
TNF Inhibitors 33
IL-1 Inhibitors 34
IL-18 Inhibitors 35
Chemokine Inhibitors 36
Inhibitors of Complement 38
Inhibitors of Interferons 39
Apoptosis 40
IV

Caspases 44
Inflammatory Caspases-1, -4, -5, -11 and -12 46
Apoptotic Initiator Caspases-2, -8, -9, and -10 48
Apoptotic Effector Caspases-3, -6 and -7 49
Intrinsic Activation of Caspases: Mitochondrial Permeability Transition Pore 51
Extrinsic Activation of Caspases: Death Receptor Mediated Signaling 53
Granzyme B 56
Apoptosis in Avian Species 57
Viral Inhibitors of Apoptosis 58
Viral Inhibitors of Mitochondria: PT Pore Modulators and Bcl-2 Homologs 59
vLIPs: Inhibitors of DISC 60
Baculovirus Caspase Inhibitor: P35 61
Baculovirus Inhibitor of Apoptosis Protein (IAP) 62
Caspase Independent Induction of Apoptosis 64
Poxviruses 65
Poxvirus Lifecycle 67
Importance of Poxviruses 69
Serine Protease Inhibitors (Serpins) 70
Poxvirus Serpins 75
SERP2 76
Cytokine Response Modifier A (CrmA) 77
SPI-2 is a CrmA Homologue 80
RPV SPI-2 and CPV CrmA Equivalency 81
In Vivo Role of CrmA during CPV Infections of CAMs 84
2 MATERIALS AND METHODS 87
Virology 87
Cells 87
Viruses 87
Viral Stock Preparation and Quantification 88
Molecular Techniques 89
Polymerase Chain Reaction (PCR) 89
DNA Manipulation, Ligation and Transformation 91
Viral DNA Preparation 92
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 92
Immunoblot 93
Pulse Labeling 94
DNA Sequencing 94
Sequence Analysis and Database Search 95
Recombinant Virus Construction 95
Transfection of Infected Cells Using Liposomes 95
Selection of Recombinant Virus in the Presence of Mycophenolic Acid 96
Reverse Selection of Recombinant Virus using 6-Thioguanine 99
Construction of Recombinant RPV Expressing CrmA 99
Construction of Recombinant RPV Expressing P35 100
v

Construction of Recombinant CPV Expressing SPI-2 102
Construction of Recombinant CPV Expressing P35 103
Construction of Recombinant CPV Expressing SERP2 104
Site Directed Mutagenesis of CrmA Gene 104
Construction of CPVCrmA D303A 105
Construction of CPVAIL-ipR Virus 105
Apoptosis Assays 106
DAPI Staining of Infected Cells 106
Preparation of Infected Cell Extracts for Caspase Activity Assay 109
Ac-DEVD-AMC Cleavage Assay for Caspase Activity 109
In Vivo Infection Assays using CAMs 110
Preparation of Chicken Chorioallantoic Membrane for Infections 110
Infecting and Harvesting CAMs Ill
Measurement of Reactive Oxygen Intermediates Ill
MTT Reduction Assay 112
Virus Infectivity Assay 112
Terminal Caspase Activity Assay on Extracts from Infected CAMs 113
Construction of Plasmids Containing Chicken ProIL-ip or ProIL-18 114
Plasmid Containing Mouse ProIL-1 p 115
Quick Coupled Transcription/Translation System 115
In Vitro Cleavage Assay for Processing ProIL-ip and ProIL-18 115
3 RESULTS 117
Equivalency of SPI-2 and CrmA 117
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Recombinant Virus Construction 118
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Prevent Apoptosis in Pig Cells 119
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Inhibit Caspase Activation 124
Recombinant RPV and CPV Viruses Expressing P35 130
RPVASPI-2::P35 Induces Apoptosis in LLC-PK1 Cells 131
CPVAcrmA: :P35 Blocks Apoptotic Induction in LLC-PK1 Cells 132
Viral Protein Expression: Effect of AraC 134
Recombinant CPV Viruses Expressing SERP2 or CrmA D303A 137
Infections of LLC-PK1 Cells with CPV Recombinants: DAPI Stained Cells 138
Infections of LLC-PK1 Cells with CPV Recombinants: Ac-DEVD-AMC
Cleavage Activity 141
Recombinant CPV Infections of CAMs 144
Pock Morphology of Recombinant CPV Infected CAMs 145
All White Pocks Contain Heterophils 145
Inflammatory Pocks Show High Levels of NADPH Oxidase 148
P35 and Serp2 Restore Viral Yields from Infected CAMs 152
P35, Serp2 and CrmA Block Induction of Terminal Caspase Activity within
Infected Pocks 153
Effect of Caspase Inhibitors on Protease Activity Present in
Infected CAMs 155
Chicken ProIL-1 p Processing Activity in CAMs 159
vi

Processing of ProIL-1 (3 can be Blocked by either SERP2 or P35 within
Inflammatory Pocks 162
Processing of ProIL-18 can be Blocked by either SERP2 or P35 within
Inflammatory Pocks 164
Chicken ProIL-18 Processing Activity in CPVAcrmA Lysates is Blocked by
Caspase Specific Peptide Inhibitors 166
Caspase-3 Activity in the Presence of Chicken CAM Extracts 171
CPVAIL-1 PR Fails to Induce Inflammation on CAMs 171
4 DISCUSSION 178
Equivalency of SPI-2 and CrmA 178
Potential Applications 181
P35 Replacements of SPI-2 and CrmA 183
Function of CrmA during CPV Infections of CAMs 184
Requirement of Intact RCL for CrmA Function In Vivo 185
Regulation of Inflammation and Apoptosis by CrmA are Distinct Functions 185
Inflammation during CPV Infection may not be due to IL-1 (3 or IL-18 186
Models of Inflammatory Response on CAMs 190
Future Studies 193
LIST OF REFERENCES 196
BIOGRAPHICAL SKETCH 239
LL\
Vll

LIST OF TABLES
Table page
1 Characterization of mice deficient for different components of the IL-1 system 15
2 List of mice deficient in the IL-1 system 16
3 Taxonomy of poxviruses 66
4 List of Primers 90
5 List of plasmids constructed 107
6 List of constructed recombinant viruses 108
7 Summary of results using recombinant CPVs 177
viii

LIST OF FIGURES
Figure page
1 Signaling by TNF, IL-1R and TLR 11
2 Apoptosis 43
3 Poxvirus replication cycle 68
4 Serpins as physiological regulators 72
5 Serpin RCL 73
6 Inhibition of trypsin by alpha-1 antitrypsin 74
7 Comparison of SPI-2 and CrmA peptide sequences 83
8 Construction of recombinant RPV 120
9 Construction of recombinant CPV 121
10 Expression of SPI-2 and CrmA during RPV infections 122
11 Expression of CrmA and SPI-2 during CPV infections 123
12 Morphological characteristics of LLC-PK1 cells infected with RPV
derivatives 125
13 Morphological characteristics of LLC-PK1 cells infected with CPV
derivatives 126
14 Biochemical changes in LLC-PK1 cells infected by RPV derivatives 128
15 Biochemical changes in LLC-PK1 cells infected by CPV derivatives 129
16 Comparison of P35 expressed in RPV and CPV 133
17 Effect of AraC on P35 expression in recombinant RPV and CPV 136
18 Expression of SERP2 and CrmA D303A recombinant proteins 139
19 Morphological changes in LLC-PK1 cells infected with CPVAcrmA::SERP2
or CPVCrmA D303A 142
20 Biochemical changes in LLC-PK1 cells infected with CPVAcrmA::SERP2
or CPVCrmA D303A 143
21 Pock morphology on CAMs infected with recombinant viruses 146
22 Histological examination of pocks from infected CAMs 149
23 Inflammatory pocks show similar levels of oxidative burst 151
24 P35 and SERP2 restore viral yields from infected CAMs 154
25 P35, SERP2 and CrmA block terminal caspase activity within CEF cells 156
26 P35, SERP2 and CrmA block terminal caspase activity within infected pocks 157
27 Chicken IL-1 p processing 161
28 P35 and SERP2 function like CrmA to prevent Caspase-1 mediated
processing of proIL-1 P 163
29 P35 and SERP2 function like CrmA to prevent processing of chicken
proIL-18 in CAMs 165
30 Caspase-1 mediated processing of chicken proIL-18 is blocked by peptides 168
31 Chicken proIL-18 processing activity in CPVAcrmA::lacZ extracts is
inhibited by caspase specific peptides 169
IX

32 Caspase-3 activity in the presence of chicken extracts 172
33 Construction of CP VAIL-ipR 174
34 CPVAIL-1 PR fails to induce inflammation during CAM infections 175
x

ABBREVIATIONS
AcNPV
Autographa californica nucleopolyhedrovirus
AP-1
activator protein-1
APC
antigen presenting cell
BIR
baculovirus IAP repeat domain
BV
baculovirus
CAM
chicken chorioallantoic membrane
CEF
chicken embryo fibroblast
CpGV
Cydia pomonella granulosisvirus
CPV
cowpoxvirus
CrmA
cytokine response modifier A
CTL
cytotoxic T lymphocytes
CV-1
monkey kidney cell line
DD
death domain
DFF
DNA fragmentation factor
DNA-PK
DNA protein kinase
DISC
death receptor induced signaling complex
E.coli gpt
E.coli guanine phosphoribosyltransferase
FADD
Fas associated death domain
G-CSF
granulocyte colony stimulating factor
xi

GM-CSF
granulocyte-monocyte colony stimulating factor
HPRT
hypoxanthine guanine phosphoribosyltransferase
IAP
inhibitors of apoptosis proteins from baculovirus
ICE
interleukin 1P converting enzyme
IFN
interferon
IL
interleukin
IRAK
IL-1R associated kinase
ITA
inhibitor of T-cell apoptosis
ITR
inverted terminal repeat
JNK
Jun N-terminal kinase
kbp
kilobase pairs
LLC-PK1
pig kidney cell line
MAPK
mitogen activated protein kinase
M-CSF
monocyte colony stimulating factor
MMP
matrix metalloproteinase
MOI
multiplicity of infection
MPA
mycophenolic acid
NADP
nicotinamide adenine dinucleotide phosphate
NF-kB
nuclear factor kappa B
NGF
nerve growth factor
NIK
NF-kB inducing kinase
NK
natural killer cell
NO
nitric oxide
Xll

OpNPV
Orgyia pseudotsugata nucleopolyhedrovirus
P35
anti-apoptotic protein from baculovirus
PARP
poly(ADP) ribose polymerase
PBS
phosphate buffered saline
PCD
programmed cell death
PR-3
proteinase-3
PT
permeability transition (mitochondrial)
RPV
rabbitpoxvirus
RCL
reactive center loop
SAPK
stress activated protein kinase
SERP2
serine proteinase inhibitor-2 from myxomavirus
SDS
sodium dodecyl sulfate
SPI-1
serine proteinase inhibitor-1 from poxviruses
SPI-2
serine proteinase inhibitor-2 from poxviruses
SPI-3
serine proteinase inhibitor-3 from poxviruses
SPI-7
serine proteinase inhibitor-7 from swinepoxvirus
STO
mouse fibroblast cell line
TACE
TNF-a converting enzyme
TDS
transient dominant selection
TIR
Toll/IL-IR domain
TLR
Toll like receptor
TNF
tumor necrosis factor
TRAF
TNF receptor associated kinase
Xlll

VAR
VV
TNFR
TRADD
variola virus
vaccinia virus
tumor necrosis factor receptor
TNFR associated death domain
xiv

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ROLE OF CrmA IN REGULATING INFLAMMATION AND APOPTOSIS DURING
COWPOXVIRUS INFECTION
By
Rajkumar Nathaniel
May 2003
Chair: Richard W. Moyer
Department: Molecular Genetics and Microbiology
Poxviruses are large DNA viruses that replicate exclusively in the cytoplasm of
the infected host cell. The importance of the host inflammatory response in controlling a
poxvirus infection is supported by the fact that approximately one third of the poxvirus
genome is directed at deflecting the early host response to virus infection. Viral serine
protease inhibitors (serpins) are proteins encoded by poxviruses that interfere with the
host immune response. Cowpox virus (CPV) encodes the serpin CrmA which has been
shown to inhibit inflammation and apoptosis during virus infection. Wild type cowpox
virus forms red, hemorrhagic, non-inflammatory pocks on the chorioallantoic membranes
(CAMs) of embryonated chicken eggs whereas CPV deleted for CrmA forms white,
inflammatory pocks accompanied by lowered yield of virus. In vitro studies have shown
CrmA to be a potent inhibitor of caspase-1, as well as caspases-8 and -10. Caspase-1 is
an enzyme responsible for the activation of the pro-inflammatory cytokines IL-1 p and IL-
18 from their inactive precursors. A CrmA-mediated block in the activation of these pro
xy

inflammatory cytokines would be consistent with the role of CrmA in controlling
inflammation. CrmA also prevents apoptosis, consistent with the demonstrated inhibition
of caspases-8 and -10. The myxoma virus serpin, SERP2, has been shown to inhibit
caspases-1, -8 and -10 as does the non-serpin caspase inhibitor, P35, from baculovirus.
However, CPV recombinants where CrmA is replaced with SERP2, P35 or CrmA D303A
(mutation of the reactive center loop (RCL) which alters substrate specificity) all produce
white, inflammatory pocks on CAMs. Virus yields are restored from the P35 and SERP2
but not CrmA D303A recombinants. Pro-apoptotic caspase-3-like activity is detected
only from CPVAcrmA::lacZ and CPVCrmA D303A infections suggesting that loss of
functional CrmA leads to apoptosis and it is apoptosis, not inflammation which is
responsible for the reduction in virus yield. We did not detect caspase-1 within infected
CAMs through processing of chicken proIL-P or proIL-18, suggesting that caspase-1
mediated control of inflammation is unlikely. We show for the first time that the
regulation of apoptosis and inflammation are two distinct functions of CrmA that may not
depend solely on caspase inhibition.
xvi

CHAPTER 1
LITERATURE REVIEW
Introduction
Poxviruses are unique DNA viruses in that they replicate exclusively in the
cytoplasm of infected cells. Smallpox, caused by variola virus, is by far the most
infamous poxvirus and has been associated with human infections for about 2000 years
(102). The ability of poxviruses to evade the effectors of antiviral immunity is evidenced
by numerous poxvirus proteins that interact with the host immune system (271).
Studying the interactions of viral proteins would be useful in understanding the immune
response to a poxvirus infection and would also help in elucidating mechanisms of
immune evasion used by poxviruses. Before discussing the role of poxvirus proteins in
modulating the host immune response, it is necessary to review basic aspects of innate
immunity, mediators of inflammation, apoptosis and poxvirus biology.
Immune Systems
The mammalian immune system is divided into the innate and adaptive
components. Innate immunity provides defense against a wide variety of pathogens
without requiring prior exposure, involves an inflammatory response and takes place at
the local site of immune challenge (reviewed in 172, 293). Although innate immunity
was thought to be nonspecific, considerable advances in our understanding of the innate
immune system have made it clear that this response can also recognize self from non-
1

2
self. Innate immunity involves a pre-programmed system that includes receptors and the
signaling mechanisms that induce a full inflammatory response that results in up
regulation of proinflammatory cytokines such as tumor necrosis factor (TNF-a),
interleukin-1(3 (IL-ip), IL-6 and interferon y (IFNy). As a consequence of
proinflammatory mediator induction, cells such as macrophages, natural killer cells and
neutrophils are activated and migrate to the site of injury. In addition, increased
generation of reactive oxygen species and degranulation of cytoplasmic granules
containing lytic enzymes occur, that aid in the toxic effects associated with these cells.
The adaptive response, on the other hand, requires prior exposure and takes place
in the draining lymph nodes. In the lymph nodes professional antigen presenting cells
(APC) such as macrophages, B-cells and dendritic cells that arrive from sites of
inflammation activate naive T and B-lymphocytes which then migrate back to the site of
inflammation. Antibodies are produced by activated B-lymphocytes and function to
neutralize the pathogen itself or offer protection against toxins made by pathogens.
Additionally antibodies bound to antigen enable phagocytic cells to ingest and destroy the
antibody coated or opsonized foreign particle. Antibodies bound to antigen can also
activate plasma proteins called complement, which through a cascade of events can lyse
and destroy certain bacteria and viruses. Activating complement also aids in
phagocytosis of pathogens. While extracellular pathogens can be taken care of by the
actions of antibody and complement, the intracellular pathogens are largely destroyed by
functions of the T-lymphocyte cell mediated response.
Interactions between T-lymphocytes and cells presenting antigenic markers drive
the cell-mediated response. Cytotoxic T cells (CTL) recognize virally infected cells and

3
cause the infected cell to undergo apoptosis or cell death thus preventing viral replication
and release of progeny. Intracellular pathogens taken up by macrophages can evade lysis
by residing in vesicles that have not fused with the lysosome. A subset to T cells termed
T-Helper 1 (ThI) cells; promote activation of macrophages which allows fusion of their
lysosomes with vesicles thus destroying the invading pathogen. Protection against
extracellular pathogens mediated by antibodies also involves the function of a third
subset of T-lymphocytes termed T-helper 2 cells (Th2). Th2 cells stimulate B-cells to
proliferate and produce antibody to particular antigens. In most cases the adaptive
response confers life long immunity against re-infection by the same pathogen.
Innate Immunity and Inflammation
Inflammation is the reaction of living tissue to any form of injury. Traditionally
inflammation is defined by the Latin words calor, dolor, rubor and tumor meaning heat,
pain, redness and swelling. These physical observations of inflammation are the effects
of cytokines on local blood flow, which allows the accumulation of fluid and cells in the
inflamed area. Primary mediators of inflammation such as interleukin-1 (IL-1) and tumor
necrosis factor (TNF), which are released from injured tissue, cause dilation of blood
vessels around the area of injury. Endothelial cells lining the blood vessels swell and
partially retract allowing the leakage of fluid into the injured area. The increase in body
temperature is a host response to proinflammatory mediators that decreases viral/bacterial
replication and increases antigen presentation by APCs. Migration and extravasation of
leukocytes depend on interactions with the endothelium. Expression of adhesion
molecules such as selectins and integrins and their interaction with corresponding ligands

4
on both leukocytes and endothelial cells control the emigration of leukocytes through
blood vessels. In addition secondary inflammatory mediators such as chemotactic
factors, chemokine IL-8 for neutrophils, are induced during inflammation and serve to
attract leukocytes to areas of tissue injury.
The purpose of an inflammatory reaction is to clear infection, remove dead tissue
and allow access of the immune system to the damaged area. The main cells initially
seen at sites of inflammation are neutrophils followed by the appearance of macrophages.
Activated neutrophils and macrophages undergo the process of respiratory burst whereby
products such as hydrogen peroxide (H2O2), superoxide radical (O2) and nitric oxide
(NO) are produced. The produced reactive oxygen and nitrogen species are released into
the extracellular milieu and act as microbicidal agents. The release of oxygen radicals
and nitric oxide, as well as proteases present in neutrophilic granules such as lysozymes
and acid hydrolases, also leads to localized tissue destruction. In addition, the activation
of clotting pathways as a result of protease action from inflammatory exudates helps in
sequestering the invading pathogen from spread. Lipid mediators such as leukotrienes
(LTB4), prostaglandins and platelet activating factor (PAF) that are released by
phagocytic cells amplify the inflammatory response. PAF is chemotactic to leukocytes
and activates PMNs. Typically the injured area is replaced by tissue identical to that
which was lost. If restitution of the original tissue is not possible due to the severity of
injury, the area is replaced by fibrous, non-specific scar tissue. An acute inflammatory
process can become chronic if the injured area is continuously damaged while being
repaired.

5
Antigen present in the draining lymph nodes activates the adaptive arm of
immunity, and thus during the later stages of an inflammatory response lymphocytes are
involved. There is a delay of 4 to 7 days from the time of injury before the effects of an
adaptive response are seen. This period is sufficient for pathogens such as poxviruses to
replicate and cause systemic spread. Therefore, in such cases the innate immune
response is crucial in controlling infections during the period before an adaptive response
is seen.
Innate Immune Recognition
The mammalian innate immune response has two steps in recognizing invading
pathogens. The first involves recognition of pathogen associated molecular patterns
(PAMPs), and the second step is based on recognition of molecular markers specific for
self (172). PAMPs are conserved products of microbial metabolism that are distributed
and common among a number of pathogens. One example of a PAMP is
lipopolysaccharide (LPS) common to all gram-negative bacteria. The molecules that
recognize PAMPs are termed pattern recognition receptors (PRRs). PRRs include a
number of molecules that can be cytosolic, membrane bound and secreted in eukaryotic
cells. PRRs function to opsonize antigens, activate complement, initiate proinflammatory
and apoptotic cascades. A common PAMP associated with virus replication is the
formation of double stranded RNA that can bind and thereby activate protein kinase R
(PKR) (69). PKR phosphorylates eukaryotic initiation factor eIF-2a which serves to
block viral and cellular protein synthesis, inducing interferon responses and causes
apoptosis of infected cells (450). Double stranded RNA also activates 2-5-

6
oligoadenylate synthase (OAS) which synthesizes 2-5-oligoadenylate. The
oligonucleotide activates RNAaseL that cleaves viral and cellular RNA (210) thus
blocking viral replication and inducing apoptosis. Other intracellular PAMPs include
Nodi, a cellular protein that binds to and regulates procaspase-9, an enzyme involved in
apoptotic induction (167). Nod2, a protein expressed in monocytes, activates nuclear
factor kappaB (NF-kB), a transcription factor associated with inflammation (294). The
complete range of ligands that interact with Nod proteins is currently unknown.
Extracellular PRRs include mannan-binding lectin (MBL) that binds mannose
residues that are abundant on microbial surfaces (111). Serine proteases MASP1 and
MASP2 are activated by MBL and cleave C2 and C4 proteins, thereby initiating the
complement cascade. C-reactive protein (CRP) and serum amyloid protein (SAP)
function as opsonins by binding to bacterial phosphorylcholine (361). The macrophage
mannose receptor (MMR) facilitates the phagocytosis of bacterial and fungal pathogens
(111). Macrophage scavenger receptor (MSR) binds LPS and protects against endotoxic
shock (409). Other examples of extracellular PRRs are the Toll/Interleukin-1 family of
receptors.
Toll and Interleukin-1 Receptors
Toll and Toll-Like Receptors (TLR)
The Toll/IL-IR family of receptors are conserved from flies to mammals and play
a central role in the induction of cellular innate immune responses. Drosophila Toll
ligand has been identified as Spatzle (221). The Drosophila Toll gene product was
originally found to be required for dorso-ventral polarity (139). Toll protein was later

7
found to control anti-fungal response in flies (221). The intracellular domain of Toll was
found to be homologous to a similar region in human interleukin-1 receptor (IL-1R)
(293). Homologs to IL-1R have since been discovered, that now belong to the Toll-like
receptor (TLR) superfamily.
TLRs are transmembrane proteins that contain leucine rich repeat (LRR)
extracellular domains and Toll/IL-IR (TIR) cytoplasmic domains. The mammalian IL-1
system will be discussed in further detail later. Similarities exist between the fly Toll and
mammalian IL-1 system. Engagement of both receptors by the respective ligands results
in the activation of nuclear transcription factors. In addition to Spatzle, there also exist
adapter proteins such as Tube and a protein kinase Pelle. To date there are at least nine
Drosophila and ten mammalian TLRs known (172). In addition to the Toll ligand,
Spatzle, there are six other Spatzle-like proteins (281). TLR4, the first mammalian TLR
identified was shown to activate NF-kB and induce IL-1, IL-6 and IL-8 genes (254).
Studies with the mouse homolog of TLR4 demonstrated its role in the recognition of LPS
and gram-negative bacteria (172). TLR2 and TLR5 also recognize bacterial components
such as peptidoglycan, LPS, lipids from trypanosomes and flagellin (150, 362), (53),
(141). TLR2 binding has been shown to cause internalization of the receptor bound
ligand to the macrophage phagosome (141,426). Whether this is an exception or a rule
for most TLRs is to be seen. Experimental evidence also suggests that TLR1, 2 and 6
may function in a cooperative manner (138, 301). This suggests that heterodimerization
of receptors could in theory increase the repertoire of ligand specificities. TLR9 is
thought to reside intracellularly on lysosomes where it recognizes unmethylated CpG
motifs in bacterial DNA and induces ThI response (145).

8
The initiation of signaling by TLRs could occur directly following receptor
engagement as in the case of TLR4 (254) (Figure 1). Alternatively the ligand may need
to be processed prior to receptor interaction as seen with Drosophila Spatzle, which
requires cleavage by serine protease Easter in order to activate Toll (221). The signaling
events following TLR engagement seem to be conserved from flies to mammals.
Signaling by TLR is depicted in Figure 1. Similar to IL-1 binding IL-1R, MyD88, an
adapter molecule that contains a TIR domain, is recruited to the receptor complex where
it causes autophosphorylation of serine threonine kinase IRAK (255). Complex protein
kinase interactions result in the activation and translocation of NF-kB to the nucleus
where it induces expression of inflammatory cytokine genes. TLR2, 5, 6 and 9 activate
NF-kB, while TLR2, 4, 6 and 9 can induce the transcription factor activator protein-1
(AP-1) activity (433). TLR2 has been shown to mediate NF-kB activity via PI3 kinase,
suggesting the involvement of GTP binding proteins (18). TLR2 involvement in
apoptosis has also been demonstrated (13, 14). The fact that mutant caspase-8 could
inhibit TLR2 activity suggests the possibility of direct activation of caspases by TLR2.
The inflammatory cytokines TNF-a, IL-1 P, IL-18 and IL-8 that are upregulated by TLRs
(254) will be discussed in detail later. The identification of more signaling events
associated with TLRs will bring to light their different roles in the immune response. The
TLR system is similar to mammalian IL-1R system (293).
Interleukin-1 (IL-1)
IL-1 is one of the earliest cytokines produced in response to cellular damage or
infection. IL-1 p is the prototypical proinflammatory cytokine that belongs to the IL-1

9
family, which includes in addition IL-la and IL-1 receptor antagonist (IL-IRa) (98).
Both IL-la and IL-ip are synthesized as 31 kDa precursors that are processed to 17 kDa
mature cytokines by cysteine proteases calpain and caspase-1 respectively (55, 58, 189,
238, 413). ProIL-la is processed between residues 112 to 113 by calpain whereas proIL-
1 p is cleaved by caspase-1 first at Asp27 followed by cleavage at Aspl 16 to yield mature
cytokine (198, 395). ProIL-la also contains a nuclear localization signal at residues 79-
86 (448). Interestingly while proIL-1 p has an absolute requirement for processing to be
functional, proIL-la is biologically active as a precursor (58, 253, 270). Neither IL-la
nor *p contains signal sequence regions that would target these proteins to the secretory
pathway (99). The predominant soluble form seems to be IL-1 p, whereas IL-la is
thought to be cell associated (21). IL-Ra is a soluble protein that is able to bind IL-1
receptors but is unable to transmit a signal and is thought to control IL-1 mediated
inflammation (86).
There are two receptors identified for IL-1. The 80 kDa IL-1 receptor type I (IL-
1RI) is responsible for signal transduction (Figure 1), whereas the 60 kDa type II receptor
(IL-1RII) does not deliver any signal and therefore functions to regulate IL-1 activity by
acting as a sink (370). The IL-1RI shares 45% homology with the cytoplasmic region of
Drosophila Toll protein (121). In addition to the TIR domain, IL-1RI and II contain
three extracellular immunoglobulin domains. Initial binding of IL-1 P to the receptor is
with low affinity. Crystal structures of IL-1 RI have revealed that binding of IL-1 p to the
receptor induces a conformational change in the receptor that allows the third Ig domain
on the receptor to wrap around the ligand (357, 435). This conformational change in the

10
receptor presumably facilitates the docking of IL-1 R accessory protein (IL-lRAcP)
resulting in high affinity binding of IL-1 and the transduction of a signal.
The key signaling molecules activated are NF-kB, AP-1 and members of the
mitogen activated protein kinase (MAPK) family (166, 276, 372, 386). The initial
signaling step post receptor engagement involves the association of adapter molecule
MyD88, a protein containing both a TIR domain and a death domain (DD) (50). The
MyD88 TIR is thought to trimerize with TIR domains of IL-1 RI and IL-lRAcP in the
signaling complex, similarly to trimerization seen with the TNF-receptor (293). MyD88
recruits IL-1R associated kinases (IRAKs) -1 and -2 (282,447). Like MyD88, IRAKs
also contain a DD, and it is conceivable that the recruitment of IRAKs to the signaling
complex is via the DDs. Although IRAK is phosphorylated on activation, a catalytically
inactive molecule is still active in signal transduction, suggesting that kinase activity is
not an absolute requirement (197, 245). IRAK functions to recruit TNF receptor
associated factor (TRAF-6), which in turn is responsible for the activation of NF-kB
inducing kinase (NIK) via kinases TAB-1 and TAK-1 (291). Consequently NIK
activates a complex called the signalsome containing IkB kinases (IKK) (42). TRAF-6
can also activate the signalsome via kinases ECSIT and MEKK1 (202, 220). This
complex activation of a number of protein kinases culminates in the activation and
translocation to the nucleus of transcription factors NF-kB and AP-1 (276, 372).
Interestingly NF-kB activation has been associated with both cell death and cell
proliferation (51). In general the state of the target cell determines the cellular pathway
involved. Post receptor signaling involves the activation of kinases of the MAPK family
that results in the activation and translocation of transcription factors. Unlike IL-1 P, IL-

11
Figure 1. Signaling by TNF, IL-1R and TLR. (A) TNF binds to TNFRI and causes the
association of TRADD, which recruits RIP and TRAF2 to the receptor complex.
Activation of IKK occurs through the activation of IKK kinases or by RIP mediated
oligomerization of IKK complexes. TNFRI induces cell death when TRADD recruits
FADD and activates caspase-8. (B) IL-1 and TLR receptor complexes which include
MyD88 and IRAKs induce the ubiquitin dependent association of TRAF6. As a result
IKK kinase Takl is activated which phosphorylates and activates the IKK complex.
Abbreviations: TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, TNF receptor
associated death domain; FADD, Fas ligand associated death domain; IKK, I kappa B
kinase; NF-kB, nuclear factor kappa B; IL, interleukin; LPS, lipopolysaccharide; IKK, I
kappa B kinase; IRAK, IL-1 receptor associated kinase, Mai, MyD88 adapter-like,
TRAF, TNF receptor associated factor. From reference 42.

12
1 Ra does not bind the third Ig domain on the receptor and cannot induce conformational
change in the receptor that would allow IL-lRAcP docking (357). IL-1 mediated signal
transduction was absent in both IL-1 RE7' and IL-lRAcP'7' mice, suggesting that both the
receptor and accessory proteins were essential components for signaling (77, 126, 212).
The IL-1RII also has three Ig domains but lacks the TIR domain (252). IL-1RII binds all
three forms of IL-1 (252). The lack of the transducing cytoplasmic TIR domain indicates
that it functions as a negative regulator of IL-1 activity. Consistent with this role, IL-
1RII has very low affinity for IL-1 Ra (398). IL-1RII is expressed highly on lymphoid
and myeloid cells where it can be shed and act as a soluble decoy receptor (73). It was
presumed that proteolytic cleavage of the extracellular domains of membrane receptors
results in the soluble forms of these receptors (83). Indeed TNF-a converting enzyme
(TACE) has been shown to shed IL-1 RII from cell membranes (334). Soluble forms of
the type I receptor have also been found in circulation. Patients with rheumatoid arthritis
and sepsis have elevated levels of soluble IL-IRs indicating that these receptors function
to regulate IL-1 activity in vivo (19,125). In areas of local inflammation, tissue necrosis
can release precursor IL-1 p into the extracellular environment where it potentially can be
processed by proteases other than caspase-1. The maturation of IL-1 p mediated by
enzymes other than caspase-1 is a consideration that may be important in the results
presented in this dissertation. Both IL-1RI and II can bind precursor IL-1P and prevent
its processing, indicating the importance of soluble receptors in inflammatory fluids
(398).
The fact that biologically active IL-1P was seen in caspase-1 '/ mice suggested the
existence of other proteases that could process proIL-1P (100). This observation may be

13
important in interpreting the results of experiments presented in this thesis. ProIL-1p can
be released from cells by an unknown mechanism despite the absence of any secretory
signal sequences in the molecule (33). In addition, cell lysis can also cause the release of
cytoplasmic proIL-1 P during tissue damage (142). A number of proteases can be found
at sites of inflammation such as in synovial fluid, sites of neutrophil and macrophage
infiltration and at sites of tissue damage. Chymotrypsin and Stapylococcus aureus
protease are able to process the 31 kDa proIL-1P to biologically active fragments ranging
from 17 to 19 kDa whereas the Trypsin cleavage product is inactive (38). Cathepsin G,
collagenase and elastase can also process proIL-1 p to bioactive cytokine (143). In
addition, the importance of such mechanisms in vivo was evidenced by similar processing
capabilities seen in synovial and bronchoalveolar lavage fluids from patients with
inflammatory polyarthritis and sarcoidosis (143). The CTL molecule granzyme A, a
serine protease, can also generate biologically active IL-ip, although this cleavage occurs
at Argl20, four residues downstream of the Aspl 16 site used by caspase-1 (169). The
serine protease chymase, derived from dermal mast cells, also converts proIL-ip to an
active species, suggesting a role for the initiation of inflammation in the skin (261).
Matrix metalloproteinases (MMPs) have also been implicated in the processing of proIL-
ip (355). Stromelysin (MMP-3) and gelatinase-A (MMP-2 and -B (MMP-9) can
generate biologically active forms of IL-ip. Extended incubation of the mature product
with these enzymes caused degradation. In addition, none of these MMPs were able to
process proIL-la. Proteinase-3, a proteolytic enzyme from activated neutrophils, has
also been shown to produce IL-1 p (71). The balance of accessible proteases at sites of
local inflammation may regulate the availability of active IL-1 p and thus modulate acute

14
and chronic states of inflammation. In summary, a number of proteases other than
caspase-1 can cleave pro-IL-ip to generate the active cytokine.
A number of knockout mice models in the IL-1 system has been studied and are
listed in Tables 1 & 2. IL-la is the least well studied, and observations indicate that it is
not involved in the inflammatory responses induced by turpentine or infection with
Listeria (98, 155). IL-1 p is crucial for the development of an acute phase response and
in the induction of febrile response to inflammation (472). Quite unexpectedly IL-1 P/_
mice were hypersensitive to both IL-a and IL-1 P, an observation that so far has not been
explained (12). Mice deleted for IL-Ra were seen to spontaneously develop autoimmune
disorders that resembled rheumatoid arthritis, arterial inflammation and were shown to be
highly susceptible to LPS induced lethality (149, 156, 290). IL-1RI knockout mice failed
to induce a biological response to either IL-la or p, indicating that this receptor is
essential for all IL-1 activity (126, 212). In IL-lRAcP7' fibroblasts, binding of IL-la or
IL-IRa was only moderately reduced, whereas IL-ip binding affinity was reduced 70-
fold indicating the requirement of accessory protein for efficient receptor binding (77).
Similarly these mice failed to induce a biological response to IL-1. Disruption of the
signaling component IRAK caused reduced activation of NF-kB in fibroblasts and
attenuated responses to IL-1 in mice (410). Caspase-17' mice are deficient in processing
IL-ip (209). Other studies have shown mature IL-1 p to be released despite caspase-1
deficiency (100). Although caspase-1 is the most important protease required for IL-ip
maturation other enzymes have been implicated to perform this function. The importance
of enzymes other than caspase-1 in IL-1 p maturation may be important in certain cases
and will be discussed in relation to the results presented in this dissertation. These

Table 1 Characterization of mice deficient for different components of the EL-1 system
IU1-specific components
Components shared with IL-18
In vitro or
in vivo
Receptors
Agonist ligands
Antagonist ligands
Signalling
Processing enzyme
response to
IU1RI
IL-lRAcP
EL-lot
ID10
IDIRa
IRAK
caspase-1
LPS
Normal
ND
ND
Variable
Increased
Reduced
Reduced
Turpentine
Reduced
ND
Normal
Reduced
Increased
ND
Normal
idi
Absent
Absent
ND
Increased
ND
Reduced
ND
EL-18
ND
ND
ND
ND
ND
Reduced
ND
IJsteria
Increased
Normal
Normal
Normal
Reduced
Normal
ND
monocytogenes
susceptibility
susceptibility
susceptibility
susceptibility
susceptibility
susceptibility
Ischaemia
Reduced
ND
ND
ND
ND
ND
Reduced
/reperfusion
Allergen
Reduced
ND
ND
ND
ND
ND
ND
challenge
Pancreatitis
Reduced
ND
ND
ND
ND
ND
Reduced
Spontaneous
inflammation
No
No
No
No
Arthropathy
arteritis
No
No
From reference 98

16
Table 2. List of mice deficient in the IL-1 system.
IL-1 system component
Mouse strain (reference)
IL-1RI
C57BL/6 x 129J (126); C57BL/6 x 129/SV (212)
IL-lRAcP
C57BL/6 (77)
IL-1 a
C57BL/6x DBA/2 (155)
IL-13
C57BL/6 x DBA/2 (155); C57BL/6 x 129EV (100)
IL-1 receptor antagonist
C57BL/6 (149, 290); MF-1 (290)
IRAK
C57BL/6 (410)
Caspase-1
C57BL/6x 129(100)

17
studies underscore the importance of IL-1 as a key proinflammatory cytokine that
mediates systemic effects and rapidly activates the innate immune response.
The end result of IL-1 signaling is the expression of genes, most of which are
involved in immunity and inflammation (81). IL-1 induces expression of a number of
cell proliferation factors such as granulocyte colony stimulating factor (G-CSF),
granulocyte-monocyte colony stimulating factor (GM-CSF) and monocyte colony
stimulating factor (M-CSF). In addition IL-1 also stimulates platelet production and
increases prostaglandin E synthesis. The production of a number of cytokines involved
in generating an inflammatory response is also stimulated by IL-1 including IL-1, -2, -3, -
6, -7, -8 and TNF. IL-1 can also activate natural killer (NK) cells, T- and B-cells. IL-1 is
a potent fever-inducing agent (81). The activities of IL-1 demonstrate its role in
inducing an inflammatory response. The crucial role of IL-1 in forming part of the innate
immune response is borne out by the fact that pathogens such as poxviruses encode
molecules that interfere with IL-1 P function. The CrmA protein encoded by
cowpoxvirus is a potent inhibitor of caspase-1 thus affecting the IL-1 P maturation
process (118). In addition cowpoxvirus also encodes a (decoy) receptor that binds IL-ip
preventing interaction with cellular IL-1R (381). Both CrmA and the virus-encoded IL-
ipR molecules are the subjects of this dissertation.
Inflammatory Mediators
Tumor Necrosis Factor (TNF)
Along with IL-1, TNF is considered a primary proinflammatory cytokine.
Although mainly produced by mononuclear cells, TNF can also be produced by a variety

18
of cells including monocytes, macrophages, mast cells, epithelial cells, neutrophils, as
well as T- and B- lymphocytes (277). It exists in two forms, TNF-a and TNF-p. TNF-a
is synthesized as a membrane-bound 26 kDa protein that is proteolytically cleaved to a
soluble 17 kDa fragment at Val77 by TNF-a converting enzyme (TACE) (39, 273).
TACE is a membrane-anchored enzyme that belongs to the disintegrin-metalloprotease
family and contains an extracellular zinc-dependent protease domain. TNF-P is a 25 kDa
glycoprotein produced by lymphocytes and is also called lymphotoxin-a (130). TNF-a
and TNF-P share 50% homology at the amino acid level (443). Both of the molecules are
active only as homotrimers (24). Although TNF-a and P have similar affinities to the
TNF receptors, they differ in their ability to cause mitogenic and cytotoxic effects (319,
360).
TNF-P is more mitogenic, as its trimers are seen to be stable for several days and
were effective in inducing fibroblast growth that requires extended presence of the
cytokine (358, 360). TNF-a trimers are less stable but have a 200-fold increase in
cytotoxicity over TNF-P (360). Gene disruptions in both TNF-a and -P protected mice
from zymosan-induced organ dysfunction, indicating the involvement of these cytokines
in inflammation (436). Both TNF-a and P are able to interact with two receptors that
mediate their biological effects (152). Binding to the receptors results in a wide range of
biological effects including cell proliferation, tumor necrosis, inflammation and
apoptosis, which are dependent on, cell condition and type of associated molecules
recruited to the signaling complex. TNF-a is very effective in controlling local infection.
However, systemic release of TNF-a is detrimental to the host leading to septic shock

19
and ultimately death by multiple organ failure due to increased vascular permeability
resulting in loss of blood volume and plasma clotting proteins.
The TNF receptor, TNF-RI, has a molecular weight of 55 kDa and acts as the
main receptor type expressed by most cells (24). TNF-RI is thought to mediate most of
the proinflammatory effects of TNF-a (315). TNF receptor mediated signaling is
depicted in Figure 1. The intracellular domain of TNF-RI contains a death domain (DD)
that is required for apoptotic signaling of cell death (402). TNF-RI is always associated
with silencer of death domains (SODD), a cellular protein that functions to prevent the
inadvertent self-association of TNF-RI death domains (173). Following TNF binding to
TNF-RI, the receptor trimerizes leading to the disassociation of SODD. The adapter
protein TNF-R associated death domain (TRADD) interacts with TNF-RI via the DDs
(159). The Fas associated death domain (FADD) adapter protein that interacts with Fas
during apoptotic signaling does not directly bind TNF-RI, but can be recruited by
TRADD to the TNF-R signaling complex. (160). The association of FADD leads to the
recruitment of procaspase-8 followed by its activation and the initiation of the caspase
cascade.
The N-terminal domain of TRADD has been found to interact with the C-terminal
domain of TNF associated factor (TRAF) 2 (160). TRAF-2 plays a role in the activation
of NF-kB and Jun N-terminal kinase (JNK) by both receptors but is not involved in
apoptosis (161, 464). RIP, a cytoplasmic serine/threonine protein kinase, strongly
interacts with TRADD in the receptor-signaling complex and activates NF-kB and JNK
pathways (159, 232). However RIP disruption (RIP'/_) affects only NF-kB but not JNK

20
activation (190). Thus is seems that TNF-RI signaling of NF-kB is mediated via RIP
while TRAF-2 enables JNK activation.
TNF-RII is a 75 kDa receptor that is co-expressed with TNF-RI predominantly in
endothelial and blood cells and is involved in passing bound TNF to TNF-RI for
signaling (151, 403). In addition, TNF-RII can directly signal NF-kB activation (152).
TNF-RII has been found to associate with a number of proteins including TRAF-1,
TRAF-2, cellular inhibitor of apoptosis protein (cIAP) -1 and cIAP-2 (343). While
TRAF-1 and -2 can form heterodimers, only TRAF-2 contacts the receptor directly (345).
Similarly cIAP-1 and -2 interact indirectly with the receptor by associating with TRAF-1
and -2 via their N-terminal BIR (baculovirus IAP repeat) domain (343). Cellular IAPs
have been shown to inhibit the induction of apoptosis by interacting with caspases-3 and -
7 directly as well as target these proteases to degradation by ubiquitination (163, 346).
Cellular IAPs have also been reported to direct the ubiquitination of TRAF-2 (227). It is
unclear at present if cIAPs have any role in the activation of NF-kB directly. Expression
of TRAF-2 is sufficient to induce NF-kB and JNK activation under certain conditions
(232, 344).
A small subset of TNF binding proteins are the soluble TNF receptors that
represent the extracellular cytokine binding domains that have been shed from the cell
surface by TACE mediated proteolytic cleavage (76, 379). The soluble receptors function
to neutralize the biological activity of TNF by antagonizing the binding of cellular
receptors (94, 179, 364, 379, 406). The outcome of TNF activity depends on the
composition of the signaling complex and can either lead to the induction of apoptosis or
activation of JNK and NF-kB pathways that function to mediate cell survival (232).

21
In summary both TNF-a and -P are able to bind TNFR and signal via DD
containing proteins initiating a cascade of events leading to the induction of apoptotic cell
death whereas events leading to IkB phosphorylation results in the translocation of NF-
kB to the nucleus where it can induce transcription of genes involved in cell survival and
the proinflammatory response. In addition TNF-a also causes neutrophil and
macrophage activation and induction to synthesize nitric oxide (NO). TNF-a also
induces the synthesis of acute phase proteins such as C-reactive protein that opsonize
microbes and activate the complement pathway. Mannan-binding lectin (MBL), a PRR
that opsonizes microbes and activates complement is up regulated by TNF-a. Like IL-
1 p, TNF-a is also a fever-inducing agent. The important role of TNF, in the innate
immune system and specifically in controlling viral infections, is evidenced by pathogens
such as poxviruses encoding molecules that interfere with TNF function. Cowpoxvirus
encodes five different TNF receptor family homologs that will be discussed in more
detail later.
Interleukin-18 (IL-18)
IL-18 was originally discovered as the factor that induced interferon gamma
(IFNy) thus being critical for the control of intracellular pathogens (296). IL-18 is a
potent inducer of CTL and NK cell activity and promotes the differentiation of T cells to
the ThI phenotype (396). While the induction of IL-18 is not entirely clear, it is seen to
be involved in an immune prophylactic response to a number of bacterial and viral
infections (35, 318). Like proIL-1 p, proIL-18 does not contain a signal peptide sequence,
is intracellular and is processed by caspase-1 (122, 133). The precursor is synthesized as

22
a 24 kDa peptide that is cleaved at Asp36 to generate the mature 18 kDa cytokine (7).
There exist alternative processing mechanisms that will be discussed later. Unlike IL-1 [3,
proIL-18 is constitutively expressed by a variety of cells including Kupffer cells,
macrophages, osteoblasts, keratinocytes, dendritic cells, astrocytes, microglial cells, T
and B cells (99, 285). A molecule purified from urine was shown to bind IL-18 (IL-
18BP) and has been the focus for potential immunotherapy (6, 292). IL-18BP was shown
to inhibit the IL-18 induction of IFN y. Interestingly a number of poxviruses also encode
a soluble vIL-18BP underlining the importance of IL-18 mediated antiviral response (52).
IL-18BP prevents IL-18 from binding its receptor and thus functions as a soluble decoy
receptor to regulate IL-18 activity.
The 60 to 100 kDa receptor for human IL-18 was originally identified as the IL-1
receptor related protein (IL-IRrp) and is now termed IL-18R/IL-18Ra has low binding
affinity for IL-18 (308, 416). Other studies have shown that T cells exhibit high affinity
binding for IL-18 suggesting the presence of a second molecule involved in the binding
complex (466). Subsequently an accessory protein-like (AcPL) receptor that is required
for high affinity binding of IL-18 and transduction of signal was identified (44).
Following binding of IL-18 to IL-18R, AcPL also termed IL-18R accessory protein
(AP)/IL-18Rp is recruited to the receptor complex where it forms a high affinity
heterodimer (336, 369). IL-18RP does not bind IL-18 by itself and does so only after IL-
18 is bound by low affinity to IL-18Ra (44). Although proteolytic processing of the
extracellular receptor domains can result in soluble forms of both molecules, only soluble
IL-18Ra (s IL-18Ra) binds IL-18 it does not neutralize the effect of IL-18. The
enhancement of affinity for IL-18 binding to IL-18Ra is supported by recent evidence

23
showing that soluble IL-18Ra only in combination with sIL-18R(3 is able to block IL-18
induction of IFNy (336). Thus the regulation of IL-18 by soluble forms of the receptors
is similar to that seen in IL-1.
The effects of receptor binding and signal transduction are similar for both IL-18
and IL-1. IL-18 signaling complex (comprising of IL-18 bound to IL-18Ra and IL-
18RP) recruits IRAK via the adapter protein MyD88 (340, 411). MyD88'/_ cells do not
respond to IL-18 (1). Upon activation, IRAK dissociates from the receptor complex and
is responsible for the activation of NIK mediated by TRAF-6 (200, 411). The signalsome
containing IkB kinases (IKK) is activated by NIK directly or alternatively by TRAF-6
activation of MEKK1 and ECSIT (42, 202, 220). The complex interaction between a
number of protein kinases culminates in the activation and translocation of transcription
factors NF-kB and AP-1 (25, 248, 340). The steps leading to the activation of NF-kB
and AP-1 seem to be identical both in IL-1 p and IL-18. IL-18 is also involved in
activating the MAPK pathway. IL-18 induced the kinase activities of MAPK and LCK in
ThI cells (419). MAPK is involvement in cell growth could possibly explain the effect
of IL-18 in activating NK and T cells (415). Apart from inducing IFNy production from
T, B and NK cells, IL-18 acts in synergy with IL-12 to induce NK cell cytotoxicity and
ThI cell differentiation (35). Signaling pathways used by IL-18 and IL-12 are quite
different. While IL-18 activates NF-kB, IL-12 activates the signal transducer and
activator of transcription (STAT) 4 (25). The lack of IFNy production by IL-18 observed
in Th2 cells may be due to the fact that TH2 cells do not express IL-18R (466). In
addition to IFNy, IL-18 is a potent inducer of the chemokines MIP-la, MCP-1, IL-8, as
well as IL-ip and TNF-a production in different cell types (321). The ability of IL-18 to

24
induce an innate response as well as stimulate the production of a number of
inflammatory cytokines underscores its importance as a proinflammatory mediator.
Both IL-18 and IL-18R have been targeted for gene disruptions. IL-18 deficient
mice have impaired IFNy production, defective NK cell activity and reduced ThI
response (399). The importance of IL-18 in controlling intracellular pathogens was
demonstrated in IL-18A mice infected with the protozoan parasite Leishmania that were
highly susceptible to infection compared to wild type controls (445). Infections with the
extracellular bacteria Stapylococcus aureus caused reduced septicemia whereas these
mice developed more severe septic arthritis, a reaction associated with impaired ThI
response (445). Receptor knockout mice have also been characterized. IL-18_/' cells
from these mice had defects in NK cytolytic activity, IFNy production and were impaired
in NF-kB or JNK activity (157). The IL-18R'/_ mice also failed to respond to IL-18
indicating the essential role of this molecule in mediating signaling.
The availability of mature IL-18 under diverse conditions can determine the
outcome of the immune response. While IL-18 is processed to maturity by caspase-1 at
Asp36, there also exist alternative processing mechanisms. In addition to caspase-1,
caspases-4 and -5 have also been shown to process proIL-18 correctly (122). Caspase-3
on the other hand processed IL-18 at Asp71 and Asp76 resulting in polypeptides with no
significant biological activity (7, 133). Proteinase-3 (PR-3), a serine protease present in
neutrophil granules has also been shown to process proIL-18 (99, 389). Both membrane
bound and soluble forms of PR-3 enzyme have been identified. PR-3 has been associated
with a number of inflammatory diseases and has recently been implicated as one the
major autoantigens in Hepatitis C virus infection (47, 99, 348, 453). Interestingly PR-3

25
can also mediate the induction of apoptosis in HL-60 cells that is independent of caspase
activity and loss of mitochondrial membrane potential in spite of being implicated in the
cleavage and activation of caspase-3 (453). Of interest will be future studies on
understanding the biological activities of PR-3 since it seems to play a role in both
inflammation and apoptosis.
In summary, IL-18 induces the production of inflammatory cytokines (TNF-a,
IL-ip and IFNy), growth factors (GM-CSF) and chemokines (IL-8, MCP-1, MIP-la and
MIP-ip). The biological activities associated with IL-18 drive a ThI response, enhance
NK cell cytotoxicity and induce macrophage activation. Thus IL-18 plays a crucial role
in the innate immune response against pathogens such as viruses. The fact that
poxviruses such as cowpoxvirus encode molecules to bind IL-18; thereby disrupting
cytokine function, indicate the importance of IL-18 as an antiviral agent and mediator of
inflammation.
Interleukin-8 (IL-8)
IL-8 was originally identified as a neutrophil chemo attractant from supernatants
of activated monocytes (359, 437, 467). IL-8 is a 7 tolO kDa protein that belongs to the
CXC family of chemotactic cytokines termed chemokines and is now called CXCL8.
Chemokines are classified based on the position of the first two N-terminal cysteines that
can be separated by one amino acid (CXC) or can be adjacent (CC) (22). Disulphide
bonds between the first and third Cys as well as the second and fourth Cys are important
for three dimensional folding, receptor recognition and biological activity. IL-8 is
synthesized as a 99 amino acid precursor that is proteolytically processed by the removal

26
of a 20 amino acid leader sequence (67). Maturation of IL-8 occurs extracellularly with a
number of proteases being implicated in the cleavage process. Two forms of mature IL-8
have been observed to be secreted from activated neutrophils IL-8 [77] and IL-8 [72]
containing 77 or 72 residues respectively which can be further processed by neutrophil
elastase, cathepsin G and proteinase-3 to give N-terminal truncated forms that are more
active (302). Neutrophil gelatinase B, a secreted matrix metalloproteinase (MMP-9) has
also been shown to cleave mature IL-8 [77] to more active truncated forms (431). Thus
inflammatory exudates containing proteases can potentiate more active forms of IL-8.
Near sites of production, chemokines can form oligomers on endothelial or
extracellular matrix, thereby forming a gradient with higher concentrations of chemokine
near the inflammatory stimulus (298). In addition to attracting neutrophils, IL-8 has also
been shown to have chemotatic effects on a number of other cells including basophils,
eosinophils, T lymphocytes and macrophages (366). The ability of IL-8 to attract
neutrophils has been disputed by both in vitro and in vivo experiments involving
intradermal injections in humans (218, 222, 405). In addition to being secreted by
neutrophils, IL-8 is also secreted by keratinocytes, fibroblasts, endothelial cells and T
cells (310). IL-8 has been demonstrated to have mitogenic effects (353). Subsequently
constitutive IL-8 expression has been observed in many types of human cancers (366).
IL-8 has also been shown to induce angiogenesis (199).
Although monomeric IL-8 is biologically active, IL-8 can also exist as a dimmer
with the disulphide bonds playing important roles in anchoring the cores of the dimmer
together (70). N-terminal residues Glu4, Leu5 and Arg6 (ELR) have been shown to be
absolutely essential for receptor binding (67, 144). The ELR motif is found to be present

27
only in chemokines that attract neutrophils (22). Synthetic IL-8 containing modifications
of the ELR sequence can function as receptor antagonists that block IL-8 action
suggesting that N-terminal truncations that occur naturally during inflamed states could
contribute to regulation (268). Two IL-8 receptors have been identified, the CXCR1 and
CXCR2 (154, 280). The receptor more selective for IL-8 is CXCR1 (269). Chemokines
exert their function via heterotrimeric seven transmembrane G-protein coupled receptors
(GPCRs) (22). The functions of the two receptors are also different in certain aspects.
While both receptors induce cytosolic free Ca2+ and degranulation, CXCR1 mediates
respiratory burst and the activation of phospholipase D (181).
Initial studies on chemokine signaling were performed in human neutrophils
stimulated with IL-8 (22, 298, 310). The functional responses were found to be similar
for all chemokine receptors. Following chemokine binding the receptor, there is a
conformational change in the chemokine at the N-terminus with disulphide bonding
between Cys7 and Cys34 (1st and 3rd Cys bond) playing a major role in receptor binding
and activation (325). The conformational change facilitates further interaction with the
receptor that leads to activation. Activation leads to the exchange of bound GDP for GTP
in the G-protein. GTP bound a subunit and Py dissociate. The signal transduction
enzymes phospholipase C (PLC) and phosphatidylinositol-3-OH kinase (PI3K) are
activated by Py subunit. PLC mediates cleavage of phosphatidylinositol (4,5)-
bisphosphate yielding inositol triphosphate (IP3) and diacylglycerol (DAG). IP3
induction results in release of intracellular stores of calcium giving rise to a transient
increase in free calcium concentration. DAG activates protein kinase C (PKC). PI3K
also generates phosphatidylinositol triphosphate and activates protein kinase B (PKB).

28
The Py subunit also brings about activation of MAP kinase and then re-associates with
GDP bound a subunits. This complex activation of kinase pathways results in the
activation of transcription factors that up regulate the expression of cell adhesion
molecules, growth factors and inflammatory cytokines that facilitate the motogenic and
mitogenic effects of IL-8.
In neutrophils, the transient increase in cytosolic free calcium and activation of
PKC is responsible for granule release and superoxide production (23). The activation of
GTPases results in cytoskeletal restructuring (40, 219). CXCR1 mediated activation of
phospholypase D also leads to superoxide formation in human neutrophils (181,211).
Activation of map kinases leads to the up regulation of transcription factors (180, 414).
Binding of ligand to CXCR2 can also lead to receptor desensitization mediated by PKC
followed by receptor endocytosis and degradation, a mechanism thought to regulate IL-8
(278, 279, 461). While the activation of transcription factors AP-1 and NF-kB by TNF-a
and IL-ip up regulate IL-8, expression of IL-8 is inhibited by interferons a and p (262,
371). Since IL-1 p and TNF-a are elevated during inflammatory states and the fact that
IL-8 is synthesized by a number of cell types, highlights its important in causing local
accumulation of neutrophils in any tissue.
In summary, IL-8 functions as a chemotactic factor for leukocytes increasing
access of immune cells such as neutrophils to sites of inflammation. IL-8 enhances
adhesion molecule binding (P2 integrin) to circulating leukocytes thereby facilitating
migration and extravasation of the immune cells across blood vessel walls into inflamed
tissue. IL-8 activates neutrophils to undergo respiratory burst and degrannulation. In

29
short, IL-8 is particularly important in the innate immune response by directing
neutrophil migration to sites of infection.
Avian Cytokines
A number of homologs for mammalian proinflammatory cytokines have been
identified in birds. A review of avian and mammalian cytokines reveals that even though
they function similarly, some of their primary structures seem to differ significantly.
Avian homologs of TNF-a, IL-1 (3, IL-18 and a number of CXC chemokines have been
identified so far. A majority of these molecules were discovered in chickens, in addition
to duck, turkey, pheasant, quail and guinea fowl (384).
TNF-a
TNF-a-like activity can be detected in chick embryos as early as day 1 (452).
The pattern of expression throughout the growing embryo suggested a role for TNF in
apoptosis during differentiation of the neuronal system as well as in tissue remodeling.
Indeed cells undergoing DNA fragmentation associated with apoptosis were
demonstrated to be closely associated with TNF-a immunoreactivity (451). Chicken
macrophages stimulated with bacterial LPS or the intracellular protozoan Eimeria,
secreted a TNF-like activity that was preferentially cytotoxic to chicken fibroblasts
indicating its role in bacterial and coccidial infections (471). Partial purification of
stimulated macrophage supernatant fractions containing TNF-like activity revealed a 17
kDa protein that cross reacted with antibodies to human TNF-a (329). Although the
partially purified protein induced biological activities related to TNF-a in chicken

30
macrophages, it was not blocked by antibodies to human TNF-a suggesting structural
differences and/or the presence of other factors in the preparation. Chicken TNF-RI has
recently been cloned and was found to be 47% identical to its human counterpart in the
cytoplasmic death domain (46). In addition a number of death receptors (DRs) related to
TNF-RI have been identified in chickens that are yet to be fully characterized (46).
IL-1
Chicken IL-1 activity was first seen in LPS stimulated spleenocyte supernatants
(140). Chicken macrophages treated with LPS also induced secretion of IL-1 activity
(41). The induction of IL-1 was seen to be dependent on protein kinase C and a
calmodulin dependent kinase. Other studies have also shown that IL-1 secretion by
macrophages is dependent on intracellular calcium levels (65). IL-1 also lowered growth
rates in chicken similar to the effects seen with inflammatory agent Sephadex (195). The
chicken IL-1 P gene was cloned rather recently and a peptide containing residues 106 to
267, the putative mature IL-p sequence, was shown to be biologically active both in vitro
and in vivo (446).
Unlike the mammalian system where maturation of IL-1 P requires caspase-1 to
process the inactive precursor initially at Asp27 (site 1) followed by cleavage at Aspl 16
(site 2), the chicken proIL-1 p sequence does not contain either of the conserved Asp
residues required for cleavage that would release the mature cytokine (395, 446). The
mechanism of IL-1 P maturation in chickens is currently unknown. It remains a
possibility that in chickens, IL-ip does not require processing and maybe active as the
precursor itself. The chicken proIL-ip sequence is similar to proIL-ip sequences from

31
frog and fish, which also do not contain the conserved Asp sites found in mammals (475).
Matrix metalloproteinases (MMPs) have also been implicated in the processing of
mammalian proIL-ip (355). Gelatinase A (MMP-2) and B (MMP-9) are among the
MMPs shown to cleave proIL-1 (3. An avian gelatinase that shares homology with MMP-
2 is observed to be over expressed in chicken embryo fibroblasts transformed with Rous
sarcoma virus (64). In addition, a chicken enzyme similar to MMP-9 has been cloned
from macrophages that differ from its mammalian counterpart in sequence and
biochemical function (137). In light of alternative mechanisms of processing observed
for mammalian IL-ip, it is worth noting that chicken heterophils treated with LPS,
zymosan or phorbol myristate acetate (PMA) show increased MMP production (328).
Although there seem to avian homologs to mammalian enzymes that have been
implicated in IL-1P maturation, there is no experimental data to prove that any of the
avian enzymes actually function to process avian IL-1 p.
The chicken IL-1RI was cloned from chicken embryo fibroblasts (136).
Interestingly the cytoplasmic region of chicken IL-1RI is nearly 80 % homologous to
both its mammalian counterpart as well as the Drosophila Toll receptor. Like other TLRs
the chicken receptor also has an extracellular Ig-like region although sequence
divergence is higher in this region. Soluble chicken IL-1RI was shown to inhibit IL-1
like activity released from LPS induced macrophages confirming an antagonistic role for
this molecule (196). The chicken homologs of IL-1 a, IL-1RII or IL-1 Receptor
antagonists are yet to be identified.

32
IL-18
The chicken IL-18 gene was discovered as a result of BLAST search analysis of a
bursal expression sequence tag (EST) database (354). Comparison of the primary
sequence between the chicken and mammalian homologs revealed that the chicken
precursor contains the conserved Asp36 that is required for processing by caspase-1 (7).
A putative peptide representing residues 30 to 198 expressed with an N-terminal
histadine tag was found to induce IFNy synthesis in primary chicken spleen cells. The
IL-18 receptor and accessory signaling protein as well as the antagonist IL-18 binding
protein are yet to be identified in birds.
CXC chemokines
The chicken chemotactic and angiogenic factor (cCAF) is a 9 kDa CXC
chemokine that was originally cloned from Rous sarcoma virus transformed fibroblasts
(29, 388). Recent studies have suggested it to be a homolog of IL-8 (183). Similar to IL-
8, the chicken cytokine can also be post translationally cleaved (by plasmin) to smaller
fragments (243). Experimental evidence points to the C-terminus as being angiogenic
(243). K60 is another chicken CXC chemokine that shares 50% homology to mammalian
IL-8 (367). K60 is secreted from macrophages stimulated with LPS, IL-ip and IFNy
(367). Interestingly both cCAF and K60 are the only chicken chemokines to date that
contain the conserved ELR motif present in all mammalian chemokines that attract
neutrophils (22). A homolog of mammalian CXCRI was cloned from a chicken genomic
library (226). The chicken receptor was found to be 67% homologous to human CXCRI.
The identification of mammalian homologs of IL-8 and related CXC chemokines in birds
demonstrate the similarities between the chemotatic systems.

33
Viral Inhibitors of Inflammation
The outcome of viral infections depends on both host and viral factors. The early
resolution of infection is the goal of the mounted innate immune reaction. Not
surprisingly, viruses have been shown to encode a number of molecules that interfere
with the early host innate response. Viral products that specifically function to abrogate
pro inflammatory cytokines and their effects will be discussed here. Cytokine synthesis is
a typical early host response to viral infection. Different viruses target the same cytokine
pathways underlining their importance as potent anti-viral agents and these include TNF-
a, IL-lp, IL-18, the interferons, complement as well as a number of chemokines. The
strategies employed by viruses function to disrupt cytokine transcription/up regulation,
interfere with receptor binding and modulate cytokine signaling.
TNF Inhibitors
Encoding decoy receptors that bind TNF is a mechanism employed by a number
of poxviruses and herpesviruses. Shope fibroma virus encodes the T2 protein that has
been shown to be a TNF receptor homolog that binds both TNF-a and p (249). A
homolog to the T2 protein was also found in Myxoma virus (427). Deletion of Myxoma
virus T2 had no effect on growth of the virus in tissue culture but significantly attenuated
viral lethality in infected rabbits. Cowpoxvirus encodes five different TNF receptor
family homologs CrmB, CrmC, CrmD, CrmE and a soluble CD30 homologue. The
CrmB protein shares homology to TNFRII in the ligand binding region and can bind both
TNF-a and P (162). CrmC also shares homology to TNFRII but only binds TNF-a
(375). CrmD found only in some strains of cowpoxvirus and ectromelia virus was able to

34
bind both TNF-a and (3 and block their cytolytic activity in vitro (233). In addition to
being present in cowpoxvirus, CrmE is also present in other poxviruses such as vaccinia
virus (352). The cowpox CrmE was also found to only inhibit TNF-a activity. More
recent work has shown the vaccinia CrmE protein to also bind TNF-a but not (3 (333). In
this study CrmE was found to inhibit the cytotoxic activities of human TNF-a but not
those of mouse or rat. Vaccinia strain USSR deleted for CrmE was attenuated in an
intranasal mouse model of infection. But vaccinia WR strain, engineered to either
express the USSR CrmE, or CPV CrmB or CPV CrmC or CPV CrmE genes displayed
enhanced virulence indicating the influence of viral TNFRs on disease outcome. The
CD30 molecule is a member of the TNF receptor family and is associated with Th2
responses although its exact function role is unclear at present (31). The important role
of CD30 in viral infection is evidenced by the fact that cowpoxvirus encodes a secreted
CD30 homologue. Another virus that interferes with TNF is the herpes human
cytomegalovirus that encodes UL144, which also belongs to the TNFR family (30).
While the ligand that binds UL144 is yet to be identified, its importance in viral infection
is demonstrated by the presence of antibodies to UL144 in patient sera.
IL-1 Inhibitors
Members of poxviruses also encode molecules that interfere with IL-1 (3 either in
its production or signaling. The subject of this dissertation work is the cowpoxvirus
CrmA protein that will be discussed in more detail later. Briefly, CrmA is believed to
inhibit the processing of proIL-1P by blocking the activity of the processing enzyme,
caspase-1 (331). The importance ofIL-1p involvement in viral infection is further

35
evidenced by the expression of soluble IL-1 (3 receptors encoded by poxviruses. The
vaccinia virus B15R protein based on sequence was proposed to function as an IL-1
binding protein (376). Indeed B15R was found to bind IL-1 p but not IL-1-a or IL-IRa
(8, 381). Viruses lacking B15R were found to be less virulent than wild type when
injected into weanling mice via the intracranial route. Similar experiments in mice
infected via the intranasal route with virus disrupted for B15R accelerated the illness and
mortality indicating a role for IL-1 in modulating virulence. Furthermore B15R was also
found to prevent fever caused by virus infection in mice. When the non-functional B15R
gene in Copenhagen, a virulent strain of vaccinia, was repaired, the resulting virus
infection suppressed fever and attenuated viral virulence (10). Recent evidence shows
that poxviruses can also interfere with signaling events downstream of TNFR and IL-1RI
such as NF-kB activation although observed responses seem to be specifically different
for each virus tested (295).
IL-18 Inhibitors
The central role of IL-18 in inducing IFNy that protects against bacterial and viral
infection is well documented (318). Interestingly a soluble cellular IL-18 binding protein
(IL-18BP) was identified and found to be more homologous to putative poxviral proteins
than to mammalian IL-18 receptors (292). In the cellular context IL-18BP functions as a
decoy receptor to bind IL-18 preventing its activity. Viral IL-18BPs (vIL-18BP) have
been identified in vaccinia, cowpox, ectromelia, variola and swinepox viruses (292). In
addition Molluscum contagiosum virus was found to contain three open reading frames
with homology to IL-18BP (457). Two of the Molluscum gene products MC53L and

36
MC54L bound both human and murine IL-18 with high affinity and prevented IL-18
mediated IFNy induction in KG-1 monocytic cells. IL-18 binding activity was found in
culture supernatants of a number of vaccinia strains as well as cowpox and ectromelia
viruses (377). Furthermore vIL-18BP of ectromelia virus was found to inhibit NF-kB
activation and IFNy induction in KG-1 monocytic cells in response to IL-18. Cowpox
and ectromelia viruses are rodent pathogens and not surprisingly their vIL-18BPs
preferentially bound mouse IL-18 over human IL-18 (52). Mice infected peritonealy
with ectromelia deleted for vIL-18BP had increased NK cell cytotoxic activity while the
mutant virus was found to be attenuated (43). IL-18 activity clearly is an important host
response mechanism seeing that a number of viruses block its activity.
Chemokine Inhibitors
The number of mechanisms employed by different viruses to subvert chemokine
activity indicates the importance of this response to control viral infection. Similar to
counter strategies used against cytokines, different herpes and poxviruses encode
molecules that regulate chemokines. Typically these fall into three categories. First are
molecules that are ligands or viral homologs of cellular chemokines. These include
human herpes virus proteins such as vMIP-I, vMIP-II, vMIP-III, that are homologs to
MIP-I a or p and function as agonists or antagonists (213, 264, 385). Cytomegalovirus
proteins vCXC-1 and -2 as well as Mareks disease virus vIL-8 are homologs of IL-8
(307, 312). Interestingly vIL-8 was found to chemoattract blood mononuclear cells but
not heterophils. Molluscum encodes two proteins that act as antagonists to MCP-1. The

37
MC148R molecule is able to bind receptors but does not transmit signal as it lacks a
region critical for receptor activation (78, 206).
The second group of viral proteins is chemokine receptor homologs. Virus
encoded chemokine receptor homologs are typically cell membrane bound and function
to bind chemokines. These include the ORF74 protein found in a number of herpes
viruses that binds CXC chemokines (342). The US28 gene of cytomegalovirus is a
functional CC chemokine receptor (36). In addition this virus also encodes US27, UL33
and UL78 proteins with homology to CC chemokine receptors some of which have been
found to localize on viral membranes (109, 240, 330). Swinepox virus encodes the K2R
molecule that is potential receptor of CXC chemokines (246) (Traci Ness and Richard
Moyer unpublished). Capripoxvirus also contains a putative CC chemokine receptor the
Q2/3L ORF (54). Molluscum encodes MC148, a MIP-ip homolog that was shown to
antagonize the chemotactic activity of a number of CC and CXC chemokines on different
cell types (78).
The third mechanism employed by viruses is to secrete binding proteins that
sequester chemokines and prevent chemotaxis of inflammatory cells to the site of
infection. Examples of these would include the myxomavirus M-T7 protein that was
originally discovered as an IFNy receptor but has subsequently been found to also bind C,
CC and CXC chemokines (214, 429). The M-Tl gene product of myxomavirus and
rabbitpoxvirus secreted 35 kDa proteins function similarly in binding CC chemokines
with high affinity and IL-8 (CXC) with a lower affinity but share limited homology (129,
216). In vivo studies indicate that M-Tl deletions in myxoma do not affect virulence but
the absence of M-Tl increased infiltrating monocytes and macrophages at the site of

38
primary infection (215). More recently M-Tl was found to differ from rabbitpoxvirus 35
kDa protein in that M-Tl can localize to cell surfaces through interactions at the C-
terminus of the molecule and heparin (363). The breadth of viral modulators of
chemokines suggests that chemotaxis is an important host response to virus infection.
Inhibitors of Complement
Activation of complement pathways can itself lead to the destruction of virus-
infected cells as well as mediate the release potent inflammatory cytokines and is another
mechanism targeted by virus infections. Mammalian regulators of complement
activation function to protect bystander cells from the effects of activated complement.
These regulators are characterized by homologous motifs termed short consensus repeats
(SCRs) (335). The vaccinia complement control protein (VCP) contains four SCRs and
regulates complement by binding to C4b and C3b (204, 251). VCP acts as a cofactor to
the cellular complement regulator serine protease factor I and aids in the cleavage of C4b
and C3b to inactive components (349). The cowpox VCP homolog called
immunomodulatory protein (IMP) was also shown to be functional in inhibiting tissue
damage due to complement at the site of viral infection both in the footpad and
subcutaneous models of mice injections (158, 258). More recently the variola homolog
of VCP termed smallpox inhibitor of complement systems (SPICE) was found to be
nearly 100 fold and 6 fold more potent at inactivating human C3b and C4b respectively
than its vaccinia counterpart (341). Glycoproteins C (gC) of human herpes virus 1 and 2
have also been found to bind C3 components of complement (203). In addition the
herpesvirus saimir CCPH gene product prevents complement mediated cell damage and

39
has been found to bind complement component C3d (108). Clearly inhibiting
complement activation and lysis of infected cells allows viruses more time to replicate
and spread.
Inhibitors of Interferons
In addition to the blocking the mentioned immune mechanisms, viral products
also mediate interferon activity and will be briefly discussed here. Poxviruses encode a
number of proteins to bind both interferon type I and II. A soluble type IIFN receptor
has been identified as the vaccinia B18R open reading frame (72, 397). B18R has high
affinity for IFNa of different species, can also be found membrane bound and its absence
from vaccinia attenuated infections in the murine intranasal model. The myxoma M-T7
products are soluble type II IFN receptors that sequester IFNy (274). A unique feature of
M-T7 is that in addition to binding IFNy it also binds IL-8 and other members of the C,
CC and CXC chemokines (214). Soluble IFNy receptor homologs appear to be present in
ectromelia, cowpox and rabbitpox viruses as well (275). Indeed the vaccinia B8R protein
binds IFNy from a different species (9). Another unique molecule is the 38 kDa
glycopeptide secreted by tanapox virus (95). Unlike any other protein known to date, the
38 kDa protein binds IFNy, IL-2 and IL-5 demonstrating yet another way viral proteins
disarm multiple immune pathways with the same molecule. There also exist numerous
protein molecules encoded by different viruses that regulate the intracellular activities of
IFN that will not be discussed here. Blocking IFN and its activity is vital for virus
survival as evidenced by numerous viral strategies discovered to date.

40
Apoptosis
Apoptosis is a crucial step necessary for the clearance of infected cells and
activated neutrophils, to protect areas adjacent to the inflammatory tissue from injury.
Apoptosis or programmed cell death is a distinct mechanism by which cells die and are
dismantled in an orderly fashion (191). This process is a distinct morphological and
biochemical function that involves cell shrinkage, membrane blebbing, degradation of
proteins, chromatin condensation, DNA fragmentation followed by disintegration of the
cell into multiple membrane enclosed vesicles termed apoptotic bodies (454). The
disassembled cell is finally phagocytosis by its neighbors. This form of cell death differs
from necrosis where cells typically lyse by loss of membrane (191). Apoptosis is part of
normal homeostatic mechanism whereby cells die during embryonic development, tissue
turnover and as a means of defense against pathogens. Metazoan development is
characterized by the over production of cells followed by apoptotic induction to cull the
excess cells at later stages of development such that the relative number of cells required
to achieve proper function is maintained. This phenomenon is first characterized in the
nematode Caenorhabditis elegans by Horvitz and others (468). Processes of assembly
and disassembly constantly maintain the normal human bodys approximately 1014 cells.
It is therefore not surprising that inappropriate cell death (either too much or too little)
can give rise to disease conditions. Excessive apoptosis has been implicated in
neurodegenerative disease such as Alzheimers (242, 387) and in tissue destruction that
occurs after vascular occlusions in the brain and heart (34, 62, 286). Similarly the lack of
apoptosis contributes to the development of tumors (2) and autoimmunity such as
autoimmune lymphoproliferative syndrome in humans (107, 339, 439).

41
Apoptosis can be induced by a number of stimuli such as radiation, growth factor
depravation, receptor-mediated signaling and viral infections. There are basic pathways
by which a cell can be signaled to initiate apoptosis as shown in Figure 2. One pathway
is extrinsic, mediated via receptor-ligand interactions specifically the receptors related to
the tumor necrosis factor (TNF) and nerve growth factor (NGF) families including
TNFR1, TNFR2 and CD95/APO-l/FasR (147). These specialized cell surface receptors
are termed death receptors (DR) (147). Ligand-receptor interaction results in receptor
oligomerization that causes specific cytoplasmic proteins to be recruited to the receptor
complexes. These cytoplasmic adapter proteins such as TRADD and FADD, then
interact with a group of cellular proteases called caspases. The activation of caspases
leads to the breakdown of cellular homeostasis that culminates in cell death.
Alternatively the second or intrinsic signaling pathway involves mitochondrial
dysfunction (Fig. 2). External cues such as loss of growth factor signals converge with
internal cues such as DNA damage and trigger the permeablization of the outer
mitochondrial membrane and leakage of mitochondrial pro-apoptotic effectors into the
cytoplasm (147). In addition the cytotoxic granule, granzyme B, a serine protease
produced by CTLs and natural killer (NK) cells has been shown to directly activate
caspases as well as the cytoplasmic protein Bid which activates the mitochondrial
pathway (26, 184). The pro-apoptotic stimulus results in the release of an array of
molecules from mitochondrial membranes, which ultimately cause cellular demise that
can be dependent or independent of caspase activation. The activation of caspases is by
far the main biochemical incident responsible for programmed cell death. Thus signaling
through DRs or the disruption of mitochondrial integrity results in the induction of

Figure 2. Apoptosis. The induction of apoptosis can be signaled extrinsically involving
TNF receptors or Fas. Receptor complexes aid in the activation of initiator caspases such
as caspase-8 and -10 which in a cascade fashion ultimately in results in the activation of
terminal caspases such as caspase-3, -6 and -7. Cellular substrates that are involved in
DNA repair and homeostasis form targets for activated caspases. Apoptotic signals can
also originate from within the cell when mitochondrial integrity is compromised. Factors
normally residing within mitochondrial membranes such as cytochrome c then leak into
the cytosol and can cause activation of caspases. Mitochondrial proteins like AIF and
EndoG can induce apoptosis independent of caspase activation. Granzyme B can directly
activate caspases or can cleave cytosolic protein Bid. Cleaved Bid translocates to the
mitochondria and associates with Bax. Transcription factor p53 regulates bcl and bax
levels. Upregulation of bcl favors cell survival, whereas upregulation of bax favors cell
death. Abbreviations: Apaf-1, apoptosis associated factor-1; AIF, apoptosis inducing
factor; CASP, caspase; Cyt c, cytochrome c; DFF, DNA fragmentation factor; DNA-PK,
DNA protein kinase; FADD, Fas associated death domain; EndoG, endonuclease G;
TNFr, tumor necrosis factor receptor; TRADD, TNFR associated death domain; UV,
ultra violet


44
apoptosis. Finally, neighboring cells and/or macrophages circumvent an inflammatory
response by phagocytosing the vesicles or apoptotic bodies formed as a result of
apoptotic induction.
Caspases
Caspases are defined as cysteine proteases that cleave protein substrates after
specific aspartic acid or the PI residue (15). There are more than 42 mammalian
caspases described to date (217). Of these 12 are found in humans, caspases-1 to -10
(119), caspase-12 (105) and caspase-14 (91). There are also caspases cloned from
diverse organisms such as worms (459, 460, 468), insects (110, 168, 365, 380) and hydra
(66).
Caspases are thiol proteases in that they make use of a cysteine side chain
nucleophile to hydrolyze cleavage of the peptide bond. Like other proteases they are
produced as zymogens and maturation involves cleavage of the propeptide after certain
aspartate residues. Caspase precursors contain an N-terminal prodomain that can vary in
length from 23 to 219 amino acids followed by regions encoding a large (p20) and a
small (plO) subunit. Sometimes spacer regions can also be present between subunits that
are excised during the maturation process (90).
The association of one large and one small subunit (p20/pl0 heterodimer) gives
rise to the catalytic domain. Each active enzyme is a tetramer composed of two such
heterodimers. The quaternary structure of caspases is unique among proteases and is
described as a homodimer of heterodimers containing two active sites at opposite ends.
While the requirement for an aspartic acid residue at the PI position of the substrate is an

45
absolute, the two residues (P2 and P3) towards the amino side of PI are not absolute but
are important in having an impact on substrate cleavage. The P4 residue on the other
hand appears to be important in determining substrate specificity. Caspases are activated
in a sequential manner beginning with those that initiate the cascade and ending in the
activation of effector caspases that result in the cleavage of the so-called death substrates.
By nature of their cleavage specificity, most caspases are capable of auto processing.
The length and presence of certain motifs on caspase prodomains are related to
their function. Caspases with large prodomains are typically involved in the initiation of
the apoptotic cascade. The death effector domain (DED) found in caspases -8 and -10
interacts with the DED of signaling adapter proteins that are associated with death
receptors involved in extrinsic signaling. Another motif, the caspase recruitment domain
(CARD) found in caspases -1,-2, -4 and -9 functions in promoting interactions between
one another and with adapter molecules. Short prodomain containing caspases are
typically effector caspases and are usually activated by initiator caspases.
Based on substrate specificities, caspases have been divided into three major
groups (119). Group I caspases are called inflammatory caspases since they function to
activate proinflammatory cytokines and include caspases-1, -4, -5, -11 and -12.
Caspases belonging to Group III function as initiator caspases and include caspases -2, -
8, -9, and -10. The initiator caspases cleave and activate Group II effector caspases that
include caspases-3, -6 and -7. Based on optimal substrate sequence specificity, caspase-2
has been placed in group II and similarly caspase-6 has been placed in group III (119).
The dual role of caspases in mediating apoptosis and inflammation indicates that these

46
two mechanisms are linked and underlines the importance of caspases in the regulation of
these two cellular processes.
Inflammatory Caspases-1, -4, -5, -11 and 12
Caspase-1 is the prototypical member of this group and was the first caspase to be
characterized. It was originally described as the interleukin-1 p converting enzyme or
ICE (58, 413). In addition to processing proIL-ip to maturation, caspase-1 has also been
shown to process proIL-18 to the mature cytokine (122, 133). The human caspase-1
precursor is a 45 kDa polypeptide that is processed at 4 distinct sites. Initial cleavage
generates the 11.5 kDa prodomain while successive cleavages generate the large and
small subunits (413). The prodomain appears to be required for dimerization,
autoactivation (430) and activation of caspases-3 and -8 (404). Over expression of
caspase-1 has been linked to apoptotic induction in rat fibroblasts, mammalian COS cells
and neuronal cell lines (117,134, 260). The first caspase knockout mice created was
caspase-1 deficient mice (209, 225). The importance of caspase-1 in the maturation of
IL-ip and IL-18 is seen in the ability of caspase inhibitors to block the formation of
bioactive cytokines (194). Although mice deficient for caspase-1 are defective in
generating mature cytokines, the animals developed normally (209). There are also
reports that caspase-1'7' mice were able to release biologically active IL-ip in response to
subcutaneous injection of turpentine indicating the presence of alternate pathways for
proIL-ip processing (100). Nevertheless the importance of caspase-1 in inflammation is
supported by evidence showing that blocking caspase-1 activity or deleting the gene
rescues mice from lethal endotoxic shock (132). Caspase-1 knockout mice were also less

47
susceptible to hypoxic-ischemic brain injury suggesting the involvement of caspase-1 in
neuronal apoptosis (113, 230). While the involvement of caspase-1 in processing
proinflammatory substrates is established, the activation of caspase-1 is less well
understood.
There is evidence to show that caspase-11 is an upstream regulator and is
responsible for the activation of caspase-1 (441). Although caspase-11 is a murine
caspase, it is thought to be a homologue of human caspase-4 (60% identical) (187). Like
caspase-1, caspase-11 can be induced by lipopolysaccharide (LPS) (440). Caspase-11
knockout mice are defective in processing proIL-1 (3 (441) and apoptotic induction (187).
There is some evidence showing the activation of caspse-11 by cathepsin B (356). There
is very little information known about the functions of caspase-4 and -5(101, 186). Like
caspase-1, they have been shown to process proIL-ip (186) and proIL-18 (122).
Caspase-5 can be induced by IFNy and LPS (228) and is found to be involved in receptor
mediated apoptosis (207). Of interest is the observation that caspase-5 can cleave the cell
cycle regulator Max at the unusual cleavage site of glutamic acid (207).
Caspase-12 was first identified in rodents and found to be localized in the
endoplasmic reticulum (ER) where it is activated by excessive stress to the ER (465).
Caspase-12 has been linked to neurodegenerative disorders (205) and was shown to
activate caspase-9 (266). Experimental evidence suggests that ischemic injury may
induce the activation of caspase-12 mediated by calpain (a cysteine endopeptidase
activated by intracellular calcium) resulting in apoptosis (284). It has been demonstrated
that procaspase-12 interacts with TNF receptor associated factor 2 (TRAF2) and is
activated in response to ER stress (465). A recent report suggests that catalytic

48
functionality of human caspase-12 has been lost since it is reported to contain a number
of mutations that prevent it from being expressed as a full length protein (105).
Apoptotic Initiator Caspases-2, -8, -9, and -10
The first caspase related disease condition described in humans is autoimmune
lymphoproliferative syndrome (ALPS) type II, an autoimmune disorder linked to
mutations in the initiator caspase-10 (439). Initiator caspases represent members
involved in both pathways leading to caspase activation namely the receptor mediated
extrinsic pathway involving caspase-8 and the intrinsic mitochondrial pathway involving
caspase-9. DR induced apoptosis involves receptor ligand interactions that aggregate and
act as scaffolds for caspase activation often termed the death inducing signaling complex
(DISC) (90). The recruitment of initiator caspases to the DISC involves the DEDs in the
prodomains of caspase-8 and -10 that are involved in binding DEDs on adaptor
molecules such as FADD in the DISC (438). Similarly the CARD found in caspase-2
and -9 prodomains has been shown to associate with CARDs on adaptor molecules such
as RAIDD facilitating the recruitment of caspase-2 to the DISC (5, 88). The induced
proximity model proposes that presence of high local concentrations of procaspase-8 in
the DISC is sufficient to stimulate the autoactivation of caspase-8 (283). This
phenomenon of autoactivation has been noted for caspase-9 and -10 (382, 438). An
alternative hypothesis is that conformational changes induced by binding adaptor
molecules can cause activation of proinitiator caspases (147). Activated caspase-8 can
either cleave the terminal effector caspases-3 and -7 directly or can cleave the

49
cytoplasmic protein Bid, which in turn activates the mitochondrial pathway (223).
Similarly activated caspase-10 has been shown to cleave procaspases-3, -7 and -8 (103).
Apoptosis can also be triggered without engaging the DRs, but instead causing
mitochondria to release proapoptotic molecules into the cytoplasm. One of these is
cytochrome c, a protein involved in electron transport, that now accumulates in the
cytoplasm and binds the apoptotic protease activating factor -1 (Apaf-1), a cytoplasmic
protein that shares homology with the C.elegans ced-4 apoptosis regulator. Cytochrome
c stimulates the formation of the mitochondrial apoptosome consisting of cytochrome c,
Apaf-1 and caspase-9. The binding of procaspase-9 to oligomers of Apaf-1 occurs
through the CARD regions of these two proteins. Like caspase-8, caspase-9 also
undergoes autoactivation induced by proximity of high concentrations of the proenzymes.
Caspase-9 processes the effector caspases-3 and -7 directly and has been shown to be the
apical caspase activated in the mitochondrial pathway responsible for the activation of
caspases-2, -6, -8 and -10 (374). Convergence of the DR and mitochondrial pathways to
cell death occurs as a result of caspase-8 mediated cleavage of cytoplasmic protein Bid,
which facilitates the release of cytochrome c from the mitochondria. This is probably as
a result of a feedback amplification loop since caspase-3 mediates activation of caspases-
2, -6, -8 and -10 following activation by caspase-9 (374).
Apoptotic Effector Caspases-3, -6 and -7
Caspase-3 which was first discovered as a gene product with high homology to
the C.elegans CED-3 protein and which caused apoptosis when overexpressed in Sf-9
insect cells (104). Subsequent work showed caspase-3 to be necessary for the cleavage

50
of poly (ADP-ribose) polymerase (PARP) an enzyme involved in DNA repair that is
proteolytically cleaved at the onset of apoptosis (289, 407). Caspase-3 is considered to
be the prototypic member of the effector caspases that function as the final executioners
of the apoptotic process (90). While the effector caspases can autoactivate, it has been
suggested that upstream caspases serve as the main activators. The initiator caspases-8, -
9 and -10 have all been found to cleave and activate procaspases-3 and -7 (90), while
caspase-3 has been shown to activate caspase-6 suggesting that this protease acts
downstream of caspase-3 (148). Caspase-3 can also be activated by caspase-1 (407),
caspase-4 (185), and caspase-11 (187). The involvement of cytotoxic T cell mediated
apoptotic induction is demonstrated in the ability of the serine protease granzyme B to
directly activate caspses-3, -6 and -7 (103, 299).
Effector caspases target substrates that are essential in DNA repair pathways,
maintaining homeostasis and structural elements of cells. In the DNA repair pathway
targeted proteins include PARP, cleaved by all effector caspases, the catalytic subunit of
DNA dependent protein kinase (DNA-PKCS) and the 70 kDa protein component of U1
ribonucleoprotein (Ul-70 kDa) that are cleaved by caspse-3 (56). The characteristic
DNA ladder formation seen in isolated apoptotic nuclei can be induced by caspase-3,
which is known to directly activate caspase activated deoxyribonucease (CAD)/ DNA
fragmentation factor (DFF) (231) and also by caspse-7 (300). In addition, other
substrates include DNA topoisomerase I & II, RNA polymerase I upstream binding factor
(UBF) (57)
The induction of apoptosis gives rise to the cleavage of molecules that affect at
least three protein kinase pathways. Caspase-3 can activate the stress activated protein

51
kinase/Jun N-terminal kinase (SAPK/JNK) pathway which has been shown to up regulate
proapoptotic genes (90) (455) as well as activate protein kinase C 5 (PKC8) during
apoptosis (123). Activated cell cycle regulators such as cyclin dependent kinases (cdk)
have also been observed to be in apoptotic cells (473). The arrays of effector caspase
substrates include those that facilitate the breakdown of cellular structure. The nuclear
matrix proteins lamin A, B & C are cleaved by caspase-6 (347). All three effectors
cleave the structural nuclear mitotic apparatus protein (NuMA) (148). Cytoskeleton
proteins that are cleaved include actin, gelsoin, fodrin and keratin (373).
Intrinsic Activation of Caspases: Mitochondrial Permeability Transition Pore
The role of mitochondria in apoptosis was established following the discovery of
several mitochondrial proteins, which normally reside in the intermembrane space, but
are released into the cytoplasm following appropriate stimuli. Mitochondria have been
known for some time to be the energy centers of eukaryotic cells. Mitochondrial
transmembrane potential (Av|/m) represents the potential between ions distributed on either
side of the inner mitochondrial membrane that results in a chemical (pH) and electric
gradient needed for mitochondrial function (20). During the induction of apoptosis, Ai|/m
collapse has been found to precede the changes associated with nuclear breakdown in a
number of cell types (208). Although the events leading to Av[/m disruption and
mitochondrial damage have not yet been elucidated, it is clear that pro- and anti-apoptotic
members of the B-cell lymphoma-2 (Bcl-2) family regulate the intrinsic mitochondrial
pathway.

52
The Bcl-2 family consists of more than 12 proteins that have been divided into
three groups based on structural and functional similarities (2, 17). Members of the anti-
apoptotic first group including Bcl-2 and Bc1-xl, contain four conserved Bcl-2 homology
(BH) domains (BH1-BH4). The C-terminal hydrophobic tails localize the proteins to the
outer mitochondrial surface while the major portion of these proteins face the cytoplasm.
The second group consists of pro-apoptotic members including Bax and Bak,
characterized by three BH domains and the C-terminal hydrophobic tail. Members of the
third group are diverse and pro-apoptotic including Bid and Bik, containing only the BH3
domain. Bcl-2 family members can form homodimers, but more importantly the pro- and
anti-apoptotic members interact to form heterodimers. Large numbers of such
heterodimers can be found in the cell. Ultimately the levels of pro- versus anti-apoptotic
family members determine the fate of the cell. An excess of pro-apoptotic Bcl-2 family
members causes cell death conversely the presence of larger numbers of anti-apoptotic
members is protective.
There are a number of models to explain the mechanism of Ai|/m disruption
mediated by the formation of permeability transition pores (PT) that allow the dissipation
of inner membrane ion gradients (147). Since Bcl-2 members oligomerize, they could
potentially form channels to facilitate protein transport. Alternatively Bcl-2 family
members could interact with other proteins to form channels. The voltage dependent
anion channel (VDAC) can bind several Bcl-2 members that regulate its activity (147).
Similarly the adenine nucleotide translocator protein (ANT) has also been implicated to
play a role in Bax induced apoptosis (244). And finally it has been proposed that Bcl-2
members themselves induce the rupture of mitochondrial membranes thereby disrupting

53
A\\im and facilitating the release of mitochondrial pro-apoptotic proteins into the
cytoplasm.
The release of cytochrome c leads to formation of the mitochondrial apoptosome
in conjunction with cytoplasmic Apaf-1 and procaspase-9, thus activating caspase-9.
Second mitochondrial activator of caspases (SMAC) or direct IAP binding protein with
low pi (Diablo), is a 25 kDa pro-apoptotic polypeptide released from mitochondria (87,
434). The first four amino acids of mature SMAC/Diablo, Ala-Val-Pro-Ile (AVPI) binds
baculovirus inhibitor of apoptosis repeat (BIR) 3 domain of X chromosome encoded
inhibitor of apoptosis protein (XIAP) (59). SMAC/Diablo can also bind the BIR2
domain of XIAP, a region important for XIAP mediated inactivation of caspases-3 and -
7. Thus SMAC binding to XIAP releases active caspases-3, -7 and -9 thereby promoting
cell death. The serine protease Htra2/Omi is also released from mitochondria and has
been shown to interact with XIAP and relieve inactivation of caspases (394). Apoptosis
inducing factor (AIF) and endonuclease G (EndoG) have recently been implicated in cell
death independent of caspase activation (224, 309, 393). Thus as a result of
mitochondrial dysfunction cell death occurs that is both dependent and independent of
caspase activity.
Extrinsic Activation of Caspases: Death Receptor Mediated Signaling
Apoptotic death, an essential feature of the mammalian immune system, regulates
lymphocyte maturation, receptor repertoire selection, homeostasis and cell-mediated
immune response. This pathway of programmed cell death makes use of specialized cell
surface receptors termed the death receptors (DRs) that belong to the tumor necrosis

54
factor receptor (TNF-R) superfamily. Members of the DR family include Fas/CD95,
TNF-R1, DR3, DR4 and DR5 (188). The common feature of these molecules is the
presence of the cytoplasmic death domain (DD) that is essential for apoptotic signal
transduction. Receptor binding leads to oligomerization and formation of the death
inducing signaling complex (DISC) that allows the recruitment of intracellular adapter
proteins to the DISC.
Interaction between the death domains of the receptors and adapter proteins such
as Fas associated death domain protein (FADD) and TNF-R associated death domain
protein (TRADD) are responsible for the binding. The adaptor molecules also contain
the death effector domains (DED) that allow recruitment of procaspases-8 and -10 to the
DISC. The initiator caspases are autocatalyzed and serve as activators of the terminal
effector caspases that cleave the death substrates. The importance of adapter molecules
like FADD in apoptosis is evidenced in cell lines derived from FADD knockout mice that
fail to undergo apoptosis induced by CD95 ligation. However FADD'7' fibroblasts were
still sensitive to cell death induced by drugs that utilize the mitochondrial pathway (470).
The discovery of viral and cellular proteins that function to block the signal
transduction events downstream of the DISC gave rise to a new family of anti-apoptotic
proteins termed the FLICE inhibitory proteins (FLIPs). The viral FLIP found in certain
herpesviruses and poxviruses inhibit the recruitment of procaspase-8 to the DISC
underlining the importance of this pathway in pathogen clearance. Casper/cFLIP is the
cellular homolog of vFLIP and contains two DEDs and a defective caspase-like domain.
Casper can associate with the DEDs of FADD and caspase-8 thereby interfering with the
formation of a functional DISC. As expected cells from Casper knockout mice are highly

55
sensitive to receptor mediated apoptotic induction (462). Interestingly gene disruptions
in FADD'7', Casper'7', and caspase-8'7' cause embryonic lethality at similar stages and
display abnormalities in heart development (432, 462, 463).
Diseases evidence the importance of DR mediated cell death in the regulation of
the immune system where either too little or too much apoptosis occurs. The
lymphoproliferation mutation (lprcg) in mice is a point mutation in the CD95 DD that
abolishes apoptotic signal transduction causing symptoms similar to those seen in
systemic lupus erythematosus. Very similar symptoms are seen in generalized
lymphoproliferative disease (gld) mice where a point mutation at the carboxy terminus of
the CD95 ligand blocks ability to ligate the CD95 receptor. In both cases there is an
accumulation of aberrant T cells. In humans, autoimmune lymphoproliferative syndrome
(ALPS) type I is a dysfunction of the CD95-CD95L system showing severe
autoimmunity with non-malignant lymphadenopathy and an increase in T cells. Thus the
failure to purge self-reactive lymphocytes can cause autoimmunity. AIDS is
characterized by a depletion of CD4+ T helper cells in the peripheral blood of patients
with human immune deficiency virus (HIV). Viral regulatory proteins such as HIV-1 Tat
enter non-infected cells and causes them to be hypersensitive to apoptosis by CD95
ligation. The binding of HIV gpl20 to CD4 also increases CD4+ T cells to undergo
apoptosis. Thus receptor-mediated apoptosis seems to be a mode of CD4+ T cell death
that plays a role in the immune regulation of HIV infection.

56
Granzyme B
Cytotoxic T cells CTLs and NK cells can selectively induce apoptosis in by
binding the death receptor CD95 on target cells or alternatively can cause apoptosis via
the cytotoxic granule granzyme B (GrB). GrB, a 35 kDa protein, is a member of the
granzyme family of lymphocyte serine proteases and is the only enzyme known to date
that shares aspartate cleaving specificity with caspases. Granzymes in general are
involved in cytolysis brought about by CTLs and NK cells. There are 5 granzymes
known to date in humans -A, -B, -H, -K and -M (184) the most abundant being GrA and
GrB (320). Granzymes are able to enter the target cells via the pore forming protein
perforin. Perforin shares structural and functional homology with the complement
membrane attack complex and gets inserted into target cell membranes where it
polymerizes to form perforin pores. Granzymes are synthesized as pre-zymogen
peptides. The pre or leader sequence targets the molecule to the secretory pathway.
Cleavage of the propeptide activates the enzymes. While purified granzymes alone have
no cytotoxic effects, their enzymatic substrates are potent cytolytic agents. GrB has been
shown to cleave procaspases-3, -6, -7, -8, -9, and -10 thus activating both initiator and
effector caspases. Cleavage of the Bcl-2 family member Bid directly by GrB activates
the mitochondrial pathway in apoptosis. In addition GrB has been shown to cleave death
substrates including PARP, DNA-PKcs, and NuMA (16, 114). Thus GrB plays an
important role in the induction of apoptosis through both the intrinsic and extrinsic
pathways.

57
Apoptosis in Avian Species
Apoptosis in the avian system is less well understood compared to the information
available from the mammalian system. Both mitochondrial mediated and DR mediated
pathways of apoptotic induction have been observed in birds. Most of these studies have
been done in chicken, quail and duck. A number of avian death receptors have been
identified to date. The first avian death receptor protein TVB was identified as the
receptor for avian leucosis sarcoma viruses (49). TVB is 37% identical to human DR-5
death receptor and has been shown to function via its death domain (48, 49). The chicken
B6 (chB6) molecule, widely used as a B lymphocyte marker, does not contain any
apparent DDs, but has been shown to induce apoptosis when cross-linked with antibodies
or overexpressed in murine cells (115, 317). The chicken homolog to human DR-6 is
76% identical to its human counterpart and contains a death domain and two TRAF
binding sites (45). Elevated levels of DR-6 protein correlated with apoptotic induction in
the hen ovary (45). Characterization of partial cDNA sequences of chicken Fas and TNF-
RI have shown 37% and 42% identity to the human homologs respectively (46).
Members of the bcl2 family have also been identified in chickens. The chicken bcl-xl
homolog functions to protect hen granulosa cells from apotosis in the phosphorylated
state (177). The bcl-2 homolog Nr-13 was originally identified as an antiapoptotic
protein activated upon infection by Rous sarcoma virus (124). Purified Nr-13 is able to
bind cytochrome c with high affinity suggesting that it blocks the formation of the
mitochondrial apoptosome involving cytochrome c and Apaf-1 thereby regulating the
induction of apoptosis (265). An IAP protein has also been identified in chickens.

58
Inhibitor of T-cell apoptosis (ITA) protein contains BIR domains as well as the zinc
finger motif characteristic of IAPs (176).
To date four avian caspases have been cloned and characterized. Chicken
caspases-1, -2, -3 and -6 (174, 175, 178). There are no reports to date of purified chicken
caspases showing biochemical activity. Chicken caspase-1 while being 44% identical to
human caspase-1 has two cysteine residues (QCCRG) in the active site pentapeptide
compared to its human homolog (QACRG) that has only one. The chicken homolog of
caspase-2 is 70% identical to its human counterpart and contains the conserved QACRG
catalytic site sequence. Chicken caspase-3 and -6-like activity was noted in granulse
cells treated with the apoptotic inducer okadaic acid. Both chicken caspase-3 and -6 are
>65% identical to the human enzymes with complete conservation in the active site
region. Caspase-6 gene disruptions in a chicken cell line had no effect on the induction
of apoptosis (347). Chicken caspase-6-like its mammalian counterpart was seen to
cleave lamins A and C. Available reports of apoptosis in the avian system suggest
conservation of both DR and mitochondrial pathways. The identification of novel
molecules in the future will highlight differences and help gain a better understanding of
this process in birds.
Viral Inhibitors of Apoptosis
Viruses are obligate intracellular parasites and therefore induction of an apoptotic
reaction to infection is a highly effective host innate response that controls and limits the
spread of the invading pathogen. Viruses, in order to survive have developed multiple
ways to either neutralize or delay the cell death machinery thereby maximizing the

59
production of progeny particles. Virus infection itself generates proapoptotic signals.
Adsorption of non-infectious virus particles to the cell surface can trigger apoptotic
signaling. Avian leukosis virus enters cells via the TVB receptor, (a member of the DR
family with significant homology to human DR-5) which has been shown to transduce
apoptotic signal (48). Viral mechanisms that block both the extrinsic and intrinsic
pathways of apoptosis have been identified. The mitochondrial pathway can also be
triggered following virus infection. Viral replication cycle activates cell cycle regulators
such as p53 is thought to involve the Bcl-2 family of proteins to induce mitochondrial
dysfunction (131). Respiratory syncitial virus can activate the ER resident caspasel2
(37). The identification of viral inhibitors in both pathways of programmed cell death
indicates the importance of apoptosis in controlling a viral infection. Inhibitors of both
the intrinsic and extrinsic pathways are discussed.
Viral inhibitors of Mitochondria: PT pore modulators and Bcl-2 homologs
Recently vaccinia virus was shown to inhibit apoptotic induction mediated by
mitochondria (444). Infections of Jurkat cells with vaccinia virus deleted for caspase
inhibitor SPI-2 were found to be resistant to Fas-induced cell death, prevent the loss of
mitochondrial membrane potential and decrease cytochrome c levels as well as caspse-3
activity. The mechanism of vaccinia virus inhibition of mitochondrion mediated
apoptosis is not clearly understood but is believed to involve the ability of the virus to
modulate the permeability transition pore. The Hepatitis B virus HBx protein binds
human VDAC and inhibits apoptotic induction by altering transmembrane potential
(324). Viral mitochondria localized inhibitor of apoptosis (vMIA) encoded by

60
cytomegalovirus forms a complex with ANT and blocks permeablization of the
mitochondrial outer membrane (128).
Modulations of mitochondrial apoptotic pathways have been observed in a
number of other viruses. Epstein-Barr virus, a y herpesvirus encodes two bcl-2
homologs. The BALF-1 protein contains two BH domains and protects transfected HeLa
cells from undergoing apoptosis when induced with anti-Fas antibody (241). The
mechanism of BALF-1 function seems to be via association with proapoptotic bcl-2
members, Bax and Bak. The BHRF-1 protein is less similar to bcl-2 and bcl-xl than
BALF-1 but has shown to protect human B cell lines from apoptotic induction with
ionomycin (146, 241). The adenovirus E1B19K protein is also a bcl-2 homolog that
blocks apoptosis during virus infection (327). E1B19K does not block the activation of
caspase-8 but rather interacts with Bax causing inhibition of cytochrome c release from
mitochondria and subsequent activation of caspase-9 (313). Further characterization has
shown it to specifically block oligomerization of Bax by preventing exposure of the Bax
BH2 epitope (391,392).
vFLIPs: Inhibitors of DISC
Collectively, proteins that inhibit the activation of initiator caspases in the DISC
belong to the family of FLICE (caspase-8) inhibitory proteins (FLIPs). A common
characteristic of FLIPs is the presence of DEDs. DED containing proteins have been
located by database searches of herpes and poxvirus genomes (32). The equine herpes
virus E8 protein interacts with the prodomain of caspase-8 preventing its activation and
possibly its recruitment to the DISC. Ectopic expression of E8 protects HeLa cells from

61
TNFRI and Fas induced apoptosis (32). MCI59 of Molluscum contagiosm virus contains
two DEDs, binds both caspase-8 and FADD and blocks apoptosis induced by Fas, TNF
and TRAIL (120). Similar vFLIPs have been found in a number of herpes viruses some
of which associate with kinases of the IKB kinase complex (127, 229).
Baculovirus Caspase Inhibitor: P35
The baculovirus anti-apoptotic P35 proteins are unique in that they do not share
homologies with any other known proteins. Interestingly these proteins share no
homology to poxvirus serpins, yet they inhibit similar target proteinases. P35 was first
identified in the insect virus Autographa californica nuclear polyhedrosis virus (AcNPV)
where P35 mutants induce apoptosis in the Sf-21 insect cell line (isolated from
Spodoptera frugipedra) (68). P35 proteins from a number of baculoviruses show distinct
abilities to inhibit apoptosis (267). Most of the P35 functional data generated to date
have used ytcNPV P35. In vitro studies have implicated P35 to be a suicide substrate for
caspases (4). P35 has been shown to inhibit all three groups of mammalian caspases and
specifically caspases-1, -3, -6, -7, -8 and -10 (474). In addition P35 has also been shown
to inhibit the C.elgans CED-3, Sf caspase-1 as well as gingipain (4, 378, 459). Recently,
the crystal structures of P35 with caspase-8 an apical initiator caspase and also with
effector Sf caspase-1 have revealed clues to its function mechanisms (92, 106, 458). The
reactive site loop (RCL) of P35 associates with the molecules main p sheet core such
that the Asp residue required for protease recognition is exposed at the apex of the loop.
Following cleavage of the RCL, there forms a covalent thio-ester linkage between the
target proteinase and P35. Cleavage also induces a dramatic conformational change in

62
P35 where the N-terminal of P35 gets repositioned into the active site of the caspase
protecting the thio-ester linkage from hydrolysis. Interestingly the presence of certain
motifs in effector caspases but absent in initiator caspases explains the preference of P35
to complex with terminal rather than apical caspases. Ectopic expression of P35 has
shown it inhibits apoptosis in a number of systems including insects, nematodes and
mammalian cells where the apoptotic signals tested included virus infection, induction by
actinomycin D, induction by Fas and TNF, NGF depravation, developmental^
programmed cell death and induction by X-radiation (259). In each case, P35 was able to
either block completely or significantly delay apoptosis. In the light of these studies, the
broad range of activity ascribed to P35 is suggestive that the molecule is a pan-caspase
inhibitor interrupting the activity of a highly conserved and ubiquitous component of the
death machinery.
Baculovirus Inhibitor of Apoptosis Proteins (IAP)
The family of inhibitor of apoptosis (IAP) was first described in baculoviruses by
Lois Miller and others (75). IAPs have also been identified in yeast, worms, insects and
mammals (350). Two zinc-binding motifs are characteristic of IAPs, the first is the
baculovirus IAP repeat (BIR) and the second being a RING domain. The BIR motif can
be found in multiple copies in a given IAP, while the RING domain is typically a single
copy located at the carboxy terminal end of the molecule (350). The RING domains
function to catalyze the degradation of IAPs and target protein through ubiquitylation
processes. The BIR domains seem to be essential for IAPs to function as anti-apoptotic
agents (89). Baculovirus IAPs were discovered by virtue of their ability to block

63
apoptosis in Sf cells infected with a AcNPV P35 mutant (80, 337). The CplA? and
OpWV proteins (isolated from baculoviruses Cydia pomonella granulosisvirus and
Orgyia pseudotsugata nucleopolyhedrovirus respectively) can also inhibit apoptosis
induced by a variety of signals including induction by, actinomycin D, expression of the
reaper and Doom genes of Drosophila melanogaster and expression of ICE in a
mammalian system (153). The baculovirus IAPs mode of inhibiting the S/caspase-1
activity is thought to affect the activation of this insect caspase, implying that the
mechanism by which IAPs function is different from the P35 mode of action (365). A
number of IAP homologues have been found in insects and mammals including the
human neuronal apoptosis inhibitory protein (NAIP), cIAP-1, cIAP-2 and XIAP (350).
The mechanism by which viral IAPs act in inhibiting apoptosis is not fully understood.
Studies with the crystal structures of human XIAP has elucidated a possible mechanism
of action that could be similar for all IAPs. The BIR3 domain of XIAP is thought to aid
the cleavage of caspase-9 and directly bind the small subunit at the C-terminus of
caspase-9 inactivating it (383, 390). With respect to caspase-3 and -7, XIAP seems to
have a completely different mode of inhibition than that seen with caspae-9 (60, 164,
338). In this case BIR2 domain is involved and complexes with caspase-3 and -7. A
linker domain N-terminus to BIR2 functions to block the substrate groove in the caspase,
in a backward or opposite orientation that is different from substrate recognition
sequences making it a novel mechanism for caspase inhibition. XIAP is not a substrate
for caspase-3 or -7 but rather functions to mask the active site. It will be of interest to
know whether the viral IAPs also function in a similar manner.

64
Caspase Independent Induction of Apoptosis
Apoptosis in the absence of active caspases seems to represent a pathway
conserved in slime mold (Dictyostelium), worms (C.elegans) and mammals (234, 297).
Two proteins have been identified to date that are released from mitochondria and are
postulated to play a role in apoptosis independent of caspase activation. The first is a
novel mitochondrial cell death inducer called the apoptosis inducing factor (AIF) (393).
Mammalian AIF is a 57 kDa flavoprotein that shares homology to oxidoreductases from
vertebrates (Xenopus laevis), non-vertebrates (C.elegans), plants, fungi and bacteria
(234). It was originally discovered as the factor able to induce caspase independent
chromatin condensation and DNA fragmentation in isolated nuclei, but itself has not been
seen to exhibit DNAse activity (393). AIF can also cause permeablization of
mitochondrial membranes and induce release of cytochrome c suggesting a role in a
positive feedback loop with the apoptosome pathway (393). Deletion of AIF in mice
causes embryonic lethality (182). While cells from knockout mice remain sensitive to
apoptosis in response to serum withdrawal, they are resistant to apoptosis induced by the
oxidative stress inducer menadione (182). Since AIF is also an oxidoreductase, it is
unclear whether observations seen in KO mice were due to loss of apoptotic activity
alone or due to the elimination of oxidoreductase activity as well.
The second toxic molecule implicated in caspase independent apoptosis is
endonuclease G (EndoG), which, like AIF, is also released from mitochondria (309).
EndoG is a 30 kDa nuclease, which was originally linked to mitochondrial DNA
replication based on its location and substrate specificity (74). Unlike CAD/DFF, EndoG
is able to induce nucleosomal DNA fragmentation independent of caspase activation

65
(224). It has also been implicated in DNA fragmentation seen in DFF deficient mouse
embryonic fibroblasts following induction of apoptosis by UV-radiation and TNF
treatment (469). A homolog to EndoG in C.elegans has been identified as CPS-6, which
shows similarity in sequence, localization and biochemical properties to mammalian
EndoG (309). Mutant C.elegans deficient in CPS-6 can be rescued by mouse EndoG
(309). The involvement of EndoG in cell death indicates the presence of an apoptotic
pathway, independent of caspase activation that is conserved from worms to mammals.
Although the caspase independent pathway is not completely understood, the
identification of AIF and EndoG clearly supports the existence of an apoptotic program
parallel to caspase activation.
Poxviruses
The Poxviridae are a family of large double stranded DNA viruses that replicate in the
cytoplasm of infected cells. The taxonomy of poxviruses is based on hosts they infect and
include the two subfamilies Chordopoxvirinae containing vertebrate viruses and
Entomopoxvirinae containing the insect poxviruses (Table 2) (271). The
Chorodopoxvirinae comprises eight genera: Orthopoxvirus, Parapoxvirus, Avipoxvirus,
Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. While
there are three genera within Entomopoxvirinae, these viruses may need to be reclassified
based on recent sequence information that suggests that they may not be as closely
related as previously thought (3, 27). Both variola virus (Orthopoxvirus genus) and
molluscum contagiosum virus {Molluscipoxvirus genus) are the only poxviruses to
exclusively infect humans (271).

66
Table 3. Taxonomy of poxviruses
Genera
Members
Orthopoxvirus
Cowpox, monkeypox, buffalopox,
vaccinia, Variola, rabbitpox,
camelpox, ectromelia, raccoonpox,
taterapox, volepox
Parapoxvirus
Pseudocowpox, parapoxvirus of red
deer, bovine papular stomatitis virus,
orf, paravaccinia virus
Vertebrate
poxviruses
Avipoxvirus
Canarypox, fowlpox, j uncopox,
mynahpox, pigeonpox, quailpox,
sparrowpox, starlingpox, turkeypox
Capripoxvirus
Goatpox, sheeppox, lumpy skin
disease virus
Lepori poxvirus
Myxoma virus, rabbit fibroma virus,
hare fibroma virus, squirrel fibroma
virus
Suipoxvirus
Swinepox virus
Molluscipoxvirus
Molluscum contagiosum virus
Yatapoxvirus
tanapox virus, Yaba monkey tumor
virus
Entomopoxvirus A
Anmala cuprea, Aphodius
tasmaniae, Demodema boranensis,
Dermolepida albohirtum, Figulus
subleavis, Melolontha melolontha
Insect
Poxviruses
Entomopoxvirus B
Amsacta moorei, Acrobasis zelleri,
Arphia conspersa, Choristoneura
biennis, Choristoneura conflicto,
Melanoplus sanguinipes, Oedaleus
senigalensis, Schistocera gregaria
Entomopoxvirus C
Aedes aegypti, Camptochironomus
tentans, Chironimus luridus,
Chironomus plumosus, Chironomus
attenuatus, Goeldichironomus
haloprasimus
Unclassified
poxviruses
California harbor sealpox, cotia
virus, dolphinpox, embu virus, grey
kangaroopox, marmosetpox,
Molluscum-like poxvirus, Nile
crocodilepox, mule deerpox virus
http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm

67
By far the most infamous poxvirus is variola, the causative agent of smallpox
infections in humans. Smallpox produced a systemic disease with a generalized rash in
humans and has been documented for about 2000 years (102). Of importance is that it is
the first and only human disease to date that has been purposefully eradicated. The
concept of variolation i.e. inoculation of dried smallpox scab material into the skin to
induce mild disease and subsequent immunity was practiced since the early 10th century
(102). It was not until 1798 when Edward Jenner demonstrated that inoculation with
cowpox could protect against smallpox. This new method, vaccination, quickly gained
widespread use and eventually led the eradication of smallpox in 1977 as a result of mass
vaccination programs carried out by the World Health Organization.
Poxvirus Lifecycle
Poxvirus genomes are linear double stranded DNA molecules that are covalently
joined at the termini. The internal core region of the genome is highly conserved among
poxviruses whereas variation occurs at the ends of the genome within the inverted
terminal repeat (ITR) regions. Genes located in this region are typically not essential for
viral growth in tissue culture, but contribute to viral pathogenesis in vivo. Poxviruses are
unique in that the entire life cycle is accomplished in the cytoplasm of the infected cell
(Figure 3). The receptor that mediates viral entry is unknown at present. However viral
entry is mediated by membrane fusion that is dependent on pH. Viral gene expression
occurs in three stages. The early genes are transcribed within the viral core that contains
all the transcription machinery. These include growth factors, immune defense
molecules such as CrmA and factors required for DNA replication. Intermediate genes

68
Figure 3. Poxvirus replication cycle. The cytoplasmic infection of a cell with an
infectious poxvirus particle. Abbreviations: EEV, extracellular enveloped virus; IMV,
intracellular mature virus; IEV, intracellular enveloped virus.

69
are transcribed concomitantly with DNA replication. Intermediate gene products are
factors for late gene transcription. Late gene products include structural proteins,
enzymes and early transcription factors. The viral DNA is then packaged within the
cytoplasmic viral factories and maturation proceeds to form infectious intracellular
mature virus (IMV) particles. IMV can be enveloped by membranes derived from the
golgi apparatus to form intracellular enveloped virus (IEV) which upon exiting the cell
looses a membrane layer and is termed extracellular enveloped virus (EEV).
Importance of Poxviruses
Poxviruses are extremely efficient in evading the effectors of antiviral immunity. Many
of these mechanisms target conserved cellular pathways. The knowledge gained from the
interactions of these viral proteins would be useful in understanding the immune response
to a poxvirus infection. Such studies also help in elucidating mechanisms of immune
evasion used by the virus. Therapeutic strategies based on models generated from viral
studies could be used to design molecules to modulate immune responses that occur in
other pathological conditions. Vaccinia virus is also an excellent vector for the
expression of foreign proteins, but the virus also elicits a tremendous immune response.
Therefore while the virus has potential as a vector in gene therapy, it might be
advantageous to minimize the side effects of treatment (immuneregulation) without
compromising gene delivery. Identifying and possibly modifying the genes responsible
for the vigorous immune response can improve its use in gene therapy. Finally, though
smallpox has officially been eradicated, interest in smallpox is sustained due to its
potential use as a weapon of mass destruction in biological warfare. Smallpox is also

70
similar to another related poxvirus namely monkeypox that causes zoonotic infections of
humans producing a clinically identical disease.
Poxviruses encode a number of molecules that aid in subverting and blocking the
host immune response at various levels. As described earlier, poxviral immune evasion
proteins include those that function to block complement activation, TNF, IL-ip, IL-18,
IFNa/p signaling via either binding proteins such as the virally encoded IL-18 binding
protein or receptor homologs such as CrmB-E which bind TNF. In order to modulate
immune cell trafficking poxviruses encode proteins that bind chemokines or are
chemokine homologs. Poxviruses encode intracellular molecules to block activation of
cellular responses to viral replication, which include the double stranded RNA binding
protein E3L and K3L a homolog to cellular initiation factor eIF-2a. Also included
among the intracellular proteins believed to interact with the host immune system are
poxvirus serpins, which are members of the serine proteinase inhibitor family. The
subject of this dissertation work involves the serpin CrmA first identified in cowpox virus
and subsequently found to have homologs in all orthopoxviruses sequenced to date
including variola and vaccinia viruses. Orthopoxviruses encode a total of three serpins
SPI-1, SPI-2/CrmA and SPI-3, which will be discussed later.
Serine Protease Inhibitors (Serpins)
The serpin protein superfamily is characterized by a conserved structure and
mode of action that can be described as suicide substrate-like inhibition (368). Serpins
are ubiquitous in nature, found in animals, plants, certain viruses and more recently in
bacteria (368, 449). Based on phylogenetic analysis serpins are grouped into 16 clades

71
and 10 divergent orphans (368). Although most serpins function to inhibit serine
proteases, there exist examples of cross class inhibition such as that exhibited by the
poxviral serpin CrmA and the cellular PI-9 which can inhibit both serine and cysteine
proteases (e.g. caspases). In addition some serpins such as ovalbumin are non-inhibitory
while corticosteroid binding globulin (CBG) and thyroxine binding globulin (TBG)
function as transport proteins (165, 311). In mammals serpins are involved in regulating
a number of processes including coagulation, fibrinolysis, inflammation, complement
activation, organ development and extracellular homeostasis as shown in Figure 4.
A metastable structural conformation is extremely important for the function of inhibitory
serpins. The conserved secondary structure is composed of three (3-sheets and at least 7
(typically 9) a-helicies. The exposed reactive center loop (RCL) is a region spanning
about 20 amino acids located at the C-terminus of the molecule which serves as the
proteinase recognition site as shown in Figure 5. Within the RCL lies the PI residue that
determines target proteinase specificity. The Pl-Pl peptide bond is cleaved during
scissile bond formation with the proteinase. Initially the proteinase forms a non-covalent
Michaelis-like complex by interactions involving residues flanking the scissile bond.
Attack by the active site serine (in the case of serine proteases) on the PI residue
leads to the formation of a covalent bond between the carbonyl of the PI residue and the
protease. At this point, the RCL begins to insert itself into (3-sheet A of the serpin while
still attached to the protease. The consequence of complete loop insertion is a 70A
translocation of the target proteinase that effectively distorts its active site resulting in
inactivation of the enzyme. The example of trypsin inactivation by alpha-1 antitrypsin is
shown in Figure 6. The formed acyl intermediate is stable from hours to weeks and is

72
neurotrophic factors
PEDF (SERPINFI)
neuroserpin (SER PI Nil)
PNI (SERPINEI)
tumor cell invasion
prohormone conversion
renal development
ACT (SERPINAS)
megsin (SERPINB7)
sperm development-
PCI (SERPINAS)
coagulation
hormone transport
9
Q
9
r!cis!S8i h PCI (SERRINAS)
inflammation &
T complement activation
UIPI (SERPINAl)
alA CT (SERPINA3)
KAL (SERPINA4)
UNE! (SERPINBI)
t i ov-serpius (SERPINB*)
blood pressure regulation
AGT (SERPINAS)
7~ angiogenesis
PEDF (SERPINF I)
maspin (SERP1NB5)?
ATIII (SERPINCI)?
PAH (SERPINEI)?
T
PCI (SERPINASj
ATIII (SERPINCI)
HCFII (SERPINDI)
PAH (SERPINEI)
ECM maintenance
and remodelling
alPI (SERPINAl) PNI (SERPINE2)
OlACT (SERPINA3) HSP47 (SERPINHI)
PAll (SERPINEI) CBP2 (SERPINHI)
B cell development
Q^ v
centerin (SERPINA9) %
fibrinolysis
T
PAI2 (SERPINB2)
PAll (SERPINEI)
02AP (SERPINP2)
apoptosis
I alPI (SERPINAl)
aJACT (SERPINA3)
ov-serpms (SERPINB*)?
microbial infection
Cl Inh (SERPINGI)
ov-serpins (SERPINB*)?
Figure 4. Serpins as physiological regulators. Serpins are involved in numerous
functions both as positive and negative regulators. From reference 368.

73
Figure 5. Serpin RCL. Position of the C-terminal reactive center loop (RCL) in the
serpin molecule. PI residue within RCL determines specificity for proteinase.

74
Trypsin (active)
Trypsin (disrupted)
Figure 6. Inhibition of trypsin by alpha-1 antitrypsin. Following formation of the
serpin-proteinase complex, the scissile bond is cleaved (PI -PI). RCL insertion into the
backbone of alpha-1 antitrypsin (serpin) causes Trypsin (target proteinase) to be
translocated such that its active site is effectively distorted resulting in inactivation of the
enzyme. From Huntington, et al. Nature, 2000

75
eventually hydrolyzed releasing the trapped previously active enzyme and cleaved serpin.
Serpin-protease complexes involving serine proteases are SDS stable and can be readily
detected on reducing gels. Whereas complexes with cysteine proteases, such as with
caspases, are intrinsically unstable due the nature of thiol ester linkages that are easily
hydrolyzed and can only be detected on native, non denaturing gels.
Poxvirus Serpins
The first poxvirus serpin discovered was CPV CrmA (cytokine response modifier A). A
total of three serpins SPI-1, SPI-2/CrmA and SPI-3 are present in orthopoxviruses. SPI-1
mutants of RPV display host range restriction. SPI-1 can form complexes with cathepsin
G (263) and more recent work suggests that SPI-1 may interact with the viral DNA
replication machinery (235). Disruption of the SPI-3 gene in VV or CPV results in cell
fusion and the formation of giant syncytium (422). SPI-3 expressed in vitro can form
stable complexes with some serine proteinases including trypsin, urokinase and tissue
plasminogen activator (420). Mutational analysis reveals that serpin function is not
required for SPI-3 to inhibit cell fusion during virus infection, suggesting SPI-3 to be a
bi-functional protein, with two separate functional motifs (423).
The SERP1, SERP2 and SERP3 proteins are serpins encoded by Myxoma virus
(135, 314, 428). SERP1 is the only secreted viral serpin and in vitro experiments have
shown SERP1 to inhibit serine proteinases including human plasmin, urokinase and
tissue plasminogen activator, as well as C1 s of the complement system suggesting an
anti-inflammatory activity for this serpin (442). The biochemical properties of SERP1
are similar to SPI-3 of orthopoxviruses. Indeed in vivo studies have proven the potent

76
action of SERP1 against inflammation (237). Although SERP1 and SPI-3 share similar
inhibitory profiles in vitro, SPI-3 cannot substitute for SERP1 in myxoma virus infections
of rabbits (442). The SERP2 protein shares a 45% similarity with CrmA/SPI-2 from
Orthopoxvirus and is also functionally similar to CrmA/SPI-2. SERP2 will be discussed
in more detail later. SERP3 was recently identified from myxoma virus and despite
having significant sequence deletions, has been proposed to retain overall serpin fold
(135). Deletion of SERP3 from myxoma virus causes attenuation of disease and prevents
viral spread to secondary sites in rabbits, suggesting a role in virulence. It is not yet
known if this serpin can inhibit proteases.
Little is known about the swine poxvirus (SPV) serpin SPI-7 that contains an Asp
at PI like CrmA/SPI-2. SPI-7 does not reveal any activity against caspases tested to date,
although SPV mutants deleted for SPI-7 have shown some attenuation (Pierre Mussy &
Richard Moyer unpublished). There are 5 serpins found in the fowlpoxvirus genome, in
addition serpins have been identified in Yaba-like disease virus (YLDV) and lumpy skin
disease virus (LSDV) (250). To date none of the poxvirus serpins are essential for virus
growth in tissue culture.
SERP2
Like CrmA, the myxoma virus serpin SERP2 also contains an Asp residue at the
PI position in the RCL. SERP2 is 35% identical to CrmA, can block caspases-1 and -8
as well as granzyme B activity (424). With respect to caspase-1, SERP2 is a less
effective inhibitor (K, = 80 nM) than CrmA (K* = 4 pM). Similarly for granzyme B,
SERP2 is four times weaker (K, = 420 nM) compared to CrmA (K, = 100 nM). Deletion

77
of the SERP2 gene in myxoma virus leads to attenuation of disease as evidenced by
survival of infected rabbits (257). SERP2 has been reported to inhibit the influx of
inflammatory cells into the site of primary inoculation as well as prevent apoptosis of
infected lymphocytes in the rabbit model. While recombinant cowpox virus expressing
SERP2 instead of CrmA failed to block the induction of apoptosis in pig kidney cells,
SERP2 in the context of myxoma virus does inhibit apoptotic induction during infection
of rabbit kidney cells (424). The importance of the PI residue in SERP2 is evidenced by
the observation that mutant viruses, in which the PI Asp is changed to Ala, cause an
attenuated disease (Amy MacNeill and Richard Moyer unpublished). If SERP2 were to
function simply as a caspase inhibitor in vivo, then a recombinant myxoma virus
expressing CrmA instead of SERP2 should produce similar effects as the wt myxoma
infection. On the contrary CrmA replacements of SERP2 in myxoma virus causes only
-60% mortality with a marked difference in primary lesion pathology (Amy MacNeill
and Richard Moyer unpublished). Thus SERP2 is an important virulence factor for
myxoma and deducing its natural target would provide clues to its role during myxoma
infection as well as its function as a serpin.
Cytokine Response Modifier A (CrmA)
CrmA was the first viral serpin molecule identified (316). It is an example of a
cross class inhibitor in that it has been shown to inhibit both the serine protease granzyme
B as well as members of the caspase family of cysteine proteases (201). CrmA was first
identified based on its ability to alter lesion morphology on chicken chorioallantoic
membranes (CAM). CPV produces characteristic red, hemorrhagic, non-inflammatory

78
pocks or lesions on 11 day old CAMs. Deletion of the CrmA gene resulted in a mutant
virus that produces white, non-hemorrhagic pocks that contained infiltrating
inflammatory cells consisting mainly of heterophils (analogous to neutrophils in
mammals) (306). Thus the gene responsible for this prevention of heterophil influx was
termed cytokine response modifier-A (CrmA). White pocks also produce much less virus
compared to wt CPV and have the ability to reduce nitroblue tetrazolium (NBT)
consistent with the presence of activated chicken heterophils. Histological analysis of the
white pocks also shows a moderate heterophilic inflammation absent in wt CPV
infections. Another study has shown that CrmA prevents the formation of (14R, 15S)
dihydroxyeicosatetraenoic acid (diHETE), an arachidonic acid metabolite during CPV
infection (305). It was further suggested that these diHETE intermediates could directly
or indirectly lead to the generation of inflammatory mediators. Whether or not this is the
case during CPV infections of CAMs has not yet been established.
Around the same time it was shown that CrmA could inhibit caspase-1 (IL-
ip convertase) in vitro (331). Caspase-1 plays a major role in the maturation of proIL-ip
and proIL-18, both of which are proinflammatory cytokines (122, 133, 413). Therefore
the role played by CrmA in blocking inflammation during CPV infections of CAMs was
thought to be through inhibition of a caspase-1 mediated release of proinflammatory IL-
1P and/or IL-18. IL-18 was previously described as the factor that induces synthesis of
interferon y (IFNy). Viral proteins that function to block the activity of both IL-1 P and
IFNy provide evidence for the importance of these host responses to poxvirus infections.
In addition to controlling IL-1 p maturation through CrmA, orthopoxviruses also encode a
soluble IL-ip receptor that binds IL-ip (381). Furthermore it is this soluble secreted

79
receptor, not CrmA that serves to control fever mediated by IL-ip in infected mice (192).
Like IL-ip, the significance of IFN in poxvirus infections can be inferred because
poxviruses control IFN both extracellularly by encoding an IFN a/p and y binding
proteins and intracellularly through proteins (vaccinia E3L and K3L genes) that interfere
with signaling events which occur after receptor binding (9, 11, 79). Deletion of either
the soluble receptors or the intracellular viral proteins E3L and K3L also leads to
attenuation of the virus (28, 456).
In addition to caspase-1, CrmA inhibits caspases-4, -5, -8, -9 and -10 at
physiological levels (118). Caspases play a central role in mediating apoptosis.
Consistent with its ability to directly inhibit a number of apical caspases, CrmA has been
shown to block the induction of apoptosis by various stimuli in different systems (117,
171, 287, 332, 407,408). Caspases can also be activated by granzyme B, a serine
protease that is part of granules synthesized by CTLs and NK cells. CrmA blocks
granzyme B mediated apoptosis by directly inhibiting granzyme B (322). Although
CrmA is shown to block the activation of caspases, its role in preventing the induction of
apoptosis during CPV infection has only been demonstrated in pig kidney cells (236,
332). Furthermore, no relationship has been found between CrmA expression and virus
yield in tissue culture as both wild type CPV and CrmA deletion mutants produce similar
amounts of infectious virus (332) and CrmA is not required for viral growth in cell
culture.

80
SPI-2 is a CrmA Homologue
The 38 kDa CrmA homolog found in other orthopoxviruses is designated as
serine protease inhibitor-2 (SPI-2). SPI-2 from rabbitpox virus (RPV) is 92% identical to
CrmA and its expression profile is similar to CrmA in virus-infected cells (236). In vitro
studies show both proteins inhibit caspase-1 with similar efficiencies. In pig kidney cells
CrmA functions to prevent the induction of apoptosis during CPV infections. But wild
type RPV despite the presence of SPI-2 cannot block apoptosis in the swine cells. In
mixed infection experiments, CrmA was able to substitute for SPI-2 in RPVASPI-2
infected cells, but the converse was not true. SPI-2 in RPV could not prevent apoptotic
induction in CPVAcrmA infections. Thus, in spite of their high degree of homology and
their identical properties in vitro, SPI-2 and CrmA do not seem to be functionally
equivalent. Ectromelia virus (EV) SPI-2 shares 94% identity with other poxviral
homologs (425). Similar to CrmA expression of EV SPI-2 also protected cells from TNF
mediated apoptosis and inhibited the activity of caspases-1 and -8.
While most of the information about CrmA has been obtained by ectopic
expression or virus infection of tissue culture cells, its role in vivo is less well understood.
Intranasal infections of mice with CPV deleted for CrmA were attenuated and showed a
higher LD50 by at least one order of magnitude (412). Mice infected with a CrmA
deletion mutant also had decreased pulmonary pathology and reduced inflammation
compared to wild type CPV infections. Conversely, deletion of the CrmA homolog SPI-
2, in vaccinia virus has no effect on virulence in mice (192) despite the fact that vaccinia
SPI-2 functioned to inhibit the induction of apoptosis during virus infection and virus
infected cell extracts were able to block the processing of proIL-1 p. Mice infected via

81
the intra dermal route with vaccinia virus deleted for SPI-2, showed enhanced pathology
(417). Surprisingly the lesions produced by the SPI-2 deleted virus were significantly
larger than those produced by wild type vaccinia. Thus although SPI-2 and CrmA are
homologous proteins, some differences do exist.
RPV SPI-2 and CPV CrmA Equivalency
Previous studies have shown that LLC-PK1 pig kidney cells infected with wt
CPV maintain a typical morphological structure of the nuclei throughout the cycle of
viral infection until cell lysis occurred (332). However pig kidney cells infected with a
CPVAcrmA mutant virus showed cytopathic effects consistent with apoptotic death.
Studies with RPV infected cells showed that RPVASPI-2 mutant virus infections also
induced typical morphological characteristics associated with apoptosis (236). But more
interestingly infections with wt RPV induced apoptosis. This result was surprising due to
the fact that RPV encodes a functional SPI-2 (CrmA) protein.
Apoptotic cells manifest a number of biochemical changes that include activation
of caspase enzymes, which lead to the cleavage of nuclear proteins such as PARP and
lamin. CPVAcrmA, RPV and RPVASPI-2 infected extracts contain an activity that
cleaved PARP from its native 116 kDa form to a 85 kDa fragment characteristically seen
in cells undergoing apoptosis whereas wt CPV infected extracts failed to do so (236).
The presence of lamin A cleaving activity in the extracts mirrored the results obtained
with PARP cleavage. These biochemical assay results correlated exactly with our
observations of infected cells stained with DAPI, which stains DNA of the cell nucleus.

82
SPI-2 of RPV shares 92% amino acid homology with CrmA, as shown in Figure
7. However these two proteins differ in the reactive site loop at positions P5 and P6,
thereby not excluding the possibility that the two proteins may have different inhibitory
specificities with respect to activated caspases in apoptotic cells. It has been reported that
there is at least one example of a caspase that recognizes residues away from the PI
cleavage site as far as the P5 residue. Therefore it is possible that the differences
between SPI-2 and CrmA at P5 and P6 may in fact play a role during virus infection.
Published reports from our laboratory have shown that caspases are activated during RPV
but not CPV infections (236). In vitro experiments with purified SPI-2 and CrmA have
indicated that both the proteins could inhibit the activity of caspase-1 with similar
efficiencies. These results suggest that although the two proteins SPI-2 and CrmA
function similarly in vitro, they may not be functionally equivalent in vivo.
Swapping the two genes between the viruses allows us to examine any differences that
might be present between SPI-2 and CrmA. If SPI-2 and CrmA have distinct properties,
then one would expect an infection of LLC-PK1 cells where CrmA is expressed in place
of SPI-2 in RPV to inhibit the induction of apoptosis (unlike wt RPV infected cells). And
similarly SPI-2 expressed in place of CrmA in CPV should induce apoptosis in LLC-PK1
cells (unlike wt CPV infections). However if there were an additional factor unique to
CPV that is required in addition to CrmA to inhibit the induction of apoptosis, then
blockage of apoptotic induction would be observed only during CPV infection regardless
of SPI-2 or CrmA expression. The results of this project are presented in this thesis and
are discussed.

83
Alignment of SPI-2 and CrmA
1 mdifreiassmkgenvfispasissvltilyygangstaeqlskyvekee 50
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
1 mdifreiassmkgenvfisppsissvltilyygangstaeqlskyvekea 50
51 nmdkvsaqnisfksinkvygrysavfkdsflrkigdkfqtvdftdcrtid 100
-I 11111-111111111111111111111 11111111111:1
51 dknk...ddisfksmnkvygrysavfkdsflrkigdnfqtvdftdcrtvd 97
101 ainkcvdiftegkinplldeqlspdtcllaisavyfkakwltpfekefts 150
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
98 ainkcvdiftegkinplldeplspdtcllaisavyfkakwlmpfekefts 147
151 dypfyvsptemvdvsmmsmygkafnhasvkesfgnfsiielpyvgdtsmm 200
111111111111111111111.111111111111111111111111111.
148 dypfyvsptemvdvsmmsmygeafnhasvkesfgnfsiielpyvgdtsmv 197
201 vilpdkidglesieqnltdtnfkkwcnsleatfidvhipkfkvtgsynlv 250
I I I I I I I I I I I I I I I I I I I I I I I I I I :: I I I I I I I I I I I I I I I I I I I
198 vilpdnidglesieqnltdtnfkkwcdsmdamfidvhipkfkvtgsynlv 247
251 dtlvksgltevfgstgdysnmcnldvsvdamihktyidvneeyteaaaat 300
I III I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
248 dalvklgltevfgstgdysnmcnsdvsvdamihktyidvneeyteaaaat 297
301 svlvadcastvtnefcadhpfiyvirhvdgkilfvgrycspttnc* 346
II I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I
298 calvadcastvtnefcadhpfiyvirhvdgkilfvgrycspttn*. 342

PI
Figure 7. Comparison of SPI-2 and CrmA peptide sequences. PI position is indicated
by arrowhead. Sequence alignment was performed using GCG Gap program.

84
In Vivo Role of CrmA During CPV Infections of CAMs
Apoptosis and inflammation are both important mechanisms by which the host
limits and clears viral infections. The results from this project will help gain a better
understanding of viral pathogenesis in terms of both virus-host interactions and the ability
of virus to modulate the host immune system during an infection.
We use the infection of CAMs of embryonated eggs as a model to study the role
of CrmA on the innate immune response including inflammation and apoptosis. The 10
day old CAM is an excellent model to study an acute inflammatory response to infection.
At this stage of embryonic development there is an absence of circulating mature B or T
cells (63,247), minimal complement protein (116) and the inflammatory response due to
CPV infection is mainly mediated by heterophils (analogous to mammalian neutrophils)
and monocytes (112). In order to understand the role played by CrmA during a CPV
infection of the CAM, we addressed the following questions (i) does CrmA prevent white
pock formation by protease inhibition, (ii) if so, is the function of CrmA solely dependent
on inhibition of caspases and (iii) can serpin homologs found in different poxviruses
function similarly?
The first question was addressed by mutating the PI Asp residue in the CrmA
RCL to alanine (D303A), which should abolish serpin function with the predicted
consequence of inducing white pocks. Such a result would also mean that an intact
PI Asp is critical for CrmAs function as an inhibitor of inflammation. The second
question was addressed by replacing CrmA in CPV with other caspase inhibitors such as
P35 of baculovirus or SERP2 from myxoma virus. If the in vivo role of CrmA was to
only inhibit caspases, then P35 and SERP2 should completely replace the function of

85
CrmA in CPV. Given its properties and the fact that SERP2 also has a PI Asp in the RCL
like CrmA, one would expect this myxoma protein to be the ideal poxvirus serpin
candidate to replace CrmA in CPV thereby addressing the third question. If SERP2 is
able to fully function like CrmA in CPV, this would indicate that serpin homologs having
identical PI residues from different poxviruses (at least in this case) have similar targets
in vivo and therefore perform similar functions.
Finally, we wanted to ascertain if IL-1 P plays a role in the inflammatory response
during CPV infections of the CAM. As mentioned earlier IL-ip can be produced by a
number of cell types in response to appropriate stimuli. Orthopoxviruses can control IL-
1P by two mechanisms. Generation of the mature cytokine can be blocked intracellularly
by CrmAs inhibition of caspase-1, while extracellular mature cytokine can be blocked
by the virus encoded IL-1P receptor. We deleted the viral IL-1P receptor in CPV and
used the resulting mutant virus (CPVAIL-ipR) to infect CAMs in order to determine
whether CPVAIL-1 PR infection inhibited or induced the formation of inflammatory
pocks. If IL-1 p were to be involved in CPV infections, one would expect the deletion of
the virus encoded IL-1 P receptor to induce an inflammatory response during CPVAIL-
1PR infections.
In this study, we show that despite their high degrees of homology SPI-2 and
CrmA are not functionally equivalent. The minute differences between the two proteins
can be recognized by different viral contexts and are amplified in the resulting infections.
We show for the first time that CrmA functions as a protease inhibitor to block
inflammation in vivo. We also show that P35 and SERP2 function like CrmA to inhibit
terminal caspase activity indicative of apoptosis during CPV infections of CAMs.

86
However, neither SERP2 nor P35 can control the inflammatory cell influx within
membranes and recombinant CPV virus in which P35 or SERP2 substitute from CrmA
each produce white pocks. We also failed to detect caspase-1 activity within infected
CAMs under any conditions, which suggests that CrmA may function to inhibit
inflammation through a novel target or mechanism. The natural target for CrmA is yet to
be elucidated. The implications to these results are discussed.

CHAPTER 2
MATERIALS AND METHODS
Virology
Cells
Primary chicken embryo fibroblasts (CEF) were obtained from 11 day old
embryonated chicken eggs according to standard methods. CEFs were maintained in
Medium 199 (Life Technologies, Grand Island, NY) supplemented with 2mM glutamine,
0.1 mM non-essential amino acids, ImM sodium pyruvate, 50U/ml penicillin, 50|ig/ml
streptomycin (Mediatech, Herndon, VA) and 5% fetal bovine serum (Life Technologies).
African Green monkey kidney (CV-1, ATCC CCL-70) cell line was maintained in
Minimum Essential Medium (MEM) with Earles salts (Life Technologies) and
supplements. Pig kidney (LLC-PK1, ATCC CL-101) cells were maintained in Medium
199 (Life Technologies) and supplements.
Viruses
Wild type cowpox virus (CPV strain Brighton Red; ATCC VR-302) and
rabbitpox virus (RPV strain Utrecht, ATCC VR-157) stocks were grown in CV-1 cells.
In this study, derivatives of CPV or RPV were made in which only the coding region for
CrmA/SPI-2 was replaced such that the regulatory elements were left intact.
87

88
Viral Stock Preparation and Quantification
Confluent CV-1 cells grown in 150mm dishes were infected in 5 ml of serum-
free medium at a multiplicity of infection (MOI) of 0.01 plaque forming units (pfu) of
virus per cell for 2 hours at 37C with 5% CO2. After absorption, an additional 20 ml of
medium containing 5% serum was added to each dish. Following 3 to 5 days of infection
when the cells showed maximal cytopathic effect (CPE), the cells were scraped into the
medium and pelleted for 4 minutes at 1000 x g. Supernatants were collected and used to
prepare viral DNA as described in the next section. The cells pellets (virus stocks) were
re-susended in 0.5 ml sterile phosphate buffered saline (PBS) per dish and frozen at -
80C. The virus stock was sonicated for 1 minute before use.
Quantification of viral stocks was performed by plaque assay on CV-1 cells.
Confluent cell monolayers of cells in 6 well dishes were infected with 10-fold serial
dilutions of virus. After a 2 hour absorption period, the inoculum was removed, and the
cells were overlayed with 2 ml of a 1:1 mixture of 1% agarose (SeaKem LE; FMC
Bioproducts, Rockland, ME) and 2X medium with serum pre-heated to 42C. The
infected dishes were incubated at 37C with 5% C02 for 3 to 4 days, until viral plaques
were visible. The cells were then stained by the addition of 1 ml of 1:30 dilution of
neutral red (Life Technologies) in serum-free medium and incubating the cells for a
further period of 4 to 6 hours. The neutral red solution was removed, and the viral titers
were determined by counting the plaques.

89
Molecular Techniques
Polymerase Chain Reaction (PCR)
Primers were obtained from Genosys-Fisher Scientific (The Woodlands, TX), and
were reconstituted in water to 100 mM. PCR was performed using a PTC-100 Thermal
Controller (MJ Research Inc., Watertown, MA). Reactions were typically carried out in
100 pi volumes containing 1U of DNA polymerase (Taq polymerase; Promega, Madison,
WI or Vent polymerase; New England Biolabs, Beverly, MA), 10 pi of 1 OX reaction
buffer (supplied by the manufacturer), 2.5 mM magnesium salt (supplied by
manufacturer), 200 pM dNTPs (Amersham Pharmacia Biotech, Piscataway, NJ), DNA (1
pg viral genomic DNA or 100 ng plasmid DNA) and water. A list of primers used is
shown in Table 3.
Typical PCR conditions were as follows: initial denaturation at 94C for 2
minutes followed by 30 cycles of denaturation at 94C for 1 minute, annealing (5C less
than the lowest melting temperature (Tm) of the primers used.) for 1 minute and extension
at 72C for 1 minute (1 minute for each kilobase of amplified product). A final extension
for 10 minutes at 72C was performed following the 30 cycles of PCR. The amplified
product (10 pi) was electrophoresed on 1% agarose (SeaKem; FMC Bioproducts) in TAE
buffer (40 mM Tris-acetate, 1 mM EDTA) containing 0.5 pg/ml ethidium bromide and
visualized using short wave ultra violet light.

90
Table 3. List of Primers
Primer
Sequence (5-3)
GM17
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA
GM18
TGCCTAGAATTCCATGGTAATCGATATTGGTCGTGT
GM19
TCG AT C G A ATT C C AT GGC A ATCG ATTTT GTT GT
GM20
ATCGATCGAATTCCCGGGCATATGCCATTTTTTTTAAAAAAAAT
AGAAAAAACATG
GM21
ATCGTACGAATTCCCGGGCATATGATCACATTCTTAATATTAG
AATATTAG
GM22
TGCTACAAGCTTGATGAACACTGATTCCGCATC
FS1
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA
FS74
TACGT C ACCC GGGTT ATTT A ATT GT GTTT A AT ATT AC
FS88
C ACGACC AAT ATCGATT ACT AT GT GT GTAATTTTTCCGG
FS89
C AAC AAAATCG ATT GCC AT GTGT GT AATTTTTCCGG
FS90
AGT AATCGAT ATT GGTCGT G
FS91
GGC AATCG ATTTTGTT G
FS129
GTCAGATCCATGGTTGAATTCCGAGCTTGGCTG
FS130
AGTCAACGCCCGGGTACGCTCACAGAATTCCCG
FS275
TGTGCGCTGGTGGCAGCATGCGCATCAACAGTTACA
FS307
T ACGTCC AT GGAT ATCTTC AGGGAAA
FS308
C AGCTACCCGGGTT AT AATTAGTT GTT GGAG AGC
FS367
ACGTAACC AT GGCT GC AGAAC AAGT AG
FS368
GTCACGGATCCCGGGCTAGTTCTGGTTTTGAAC
FS401
ACGTAGCCATGGCGTTCGTTCCCGACCTG
FS402
GCATTCGGATCCTCAGCGCCCACTTAGCT
FS403
AGT CAT GAATTC AGT AT ACC ACCTGTTAT
FS408
CTGCACGGATCCCAATCACGTTATACTAATAGTAAC
FS409
GCTTACTC AT GAGCTGTG AAG AGA
FS410
ACTTCGGGATCCTGATCATAGGTTGTGCCT
FS413
ACTCCCTGCAGTCATTAATCATTATCCGCTCCTCG
FS414
TCAGACTGCAGCCCGGTAGTTGCGATATAC
FS415
TGACGCCCGGGAGATTAGCGACCGGAGATT
FS416
AGCTGCCCGGGCGTTGAGACCTCCCACAACG

91
DNA Manipulation, Ligation and Transformation
Plasmid or PCR amplified DNA was digested with restriction endonucleases
according to manufacturers instructions (New England Biolabs). Digested DNA (1 pg
of plasmid DNA or 100 pi of PCR product) was purified after agarose gel electrophoresis
using the Geneclean II kit (Bio 101, Vista, CA) according to manufacturers instructions.
The purified DNA was quantified in a TD-700 fluorimeter (Turner Designs, Sunnyvale,
CA). Vector and insert DNA mixed at 1:2 and 1:10 ratios, respectively, were ligated in
20 pi volumes using 50 to 100 ng total DNA and T4 DNA ligase (New England Biolabs)
according to manufacturers instructions. Ligations were incubated at 16C overnight.
The entire ligation mixture was transformed into electrocompetant E.coli DH5a cells as
described by Sambrook (351). Transformed colonies of E.coli cells were grown on LB
agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% Difco Bacto agar; Becton
Dickinson, Sparks, MD) containing the appropriate antibiotic for selection. Transformed
colonies were screened by colony PCR using the cells directly as templates.
Briefly, using sterile toothpicks, single colonies were picked and streaked onto
LB agar plates (containing the appropriate antibiotic for selection), the toothpick was
then immersed in PCR reaction mixture (containing PCR buffer, primers specific for the
cloned gene and enzyme) that had been previously aliquoted into PCR tubes. The PCR
reaction was then carried out as described earlier. An aliquot of the PCR product
(typically 10 pi) was electrophoresied on 1% agarose gels. The presence of the cloned
gene in transformed colonies was confirmed by visualizing the PCR product under UV
light. Alternatively transformed colonies were screened by restriction enzyme digestion

92
following isolation of plasmid DNA from cells using QIAfilter Maxi-prep kits (Qiagen,
Valencia, CA).
Viral DNA Preparation
Confluent monolayers of CV-1 cells were infected with either RJPV, CPV or their
derivatives, as described in viral stock preparation. After complete CPE, the supernatants
were collected following centrifugation of cells at 1000 x g for 4 minutes. The
supernatants were transferred to 30ml polypropylene tubes, and extracellular enveloped
virus (EEV) was pelleted by. The viral pellet was re-suspended in water (100 pi per 150
mm dish) containing 1% SDS (Sigma Chemical, St.Louis, MO) and 1 mg/ml proteinase
K (Sigma) and incubated overnight at 50C.
The solutions were mixed with an equal volume of phenol/chloroform and
centrifuged at 16,000 x g for 4 minutes. The aqueous layer was mixed with chloroform
and centrifuged at 16,000 x g for 4 minutes. DNA in the aqueous phase was precipitated
with 2 volumes of 95% ethanol and 1/10 volume of 3M sodium acetate (ph 5.3) at -20C
overnight. DNA was centrifuged at 16,000 x g for 4 minutes in microfuge tubes, and the
pellets were washed once with 70% ethanol and re-suspended in water (100 pi per
150mm dish). The DNA was quantified using a fluorimeter.
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Extracts of infected cells were prepared by resuspending cell pellets in Triton
lysis buffer (100 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% v/v Triton X-100) and lysed
by 3 freeze-thaw cycles. Cell debris was pelleted at 1500 x g for 10 minutes and the

93
supernatants were collected. Protein concentrations of supernatants were measured using
Coomasie Stain kit (Pierce Chemical Co., Rockford, IL). Proteins were resolved on
sodium deodecyl sulfate polyacrylamide gels (SDS-PAGE) by electorphoresis. Typically
10% w/v polyacrylamide gels were run using the BioRad Protean II apparatus (Bio-Rad
Laboratories, Hercules, CA). Extracts or proteins were boiled in SDS loading buffer
(62.5 mM Tris pH 6.8, 20% glycerol, 2% SDS, 5% p-mercaptoethanol and 0.025%
bromophenol blue) for 5 minutes and loaded onto a gel. Typically gels were
electrophoresed overnight at 75 volts.
Immunoblot
Proteins that had been resolved on SDS-PAGE were transferred to nitrocellulose
membranes (Micron Separations, Westborough, MA) in transfer buffer (25 mM Tris pH
8.3, 192 mM glycine, 20% methanol) using a semi-dry transfer apparatus (Fisher
Scientific, Hampton, NH) at 225 mAmps for 2 hours. Membranes were blocked in
blocking buffer (5% w/v nonfat milk powder in PBS pH 7.4, 0.1% v/v Tween 20) for 1
hour at room temperature. Membranes were probed with primary antibody at the
appropriate dilution in blocking buffer for at least 1 hour at room temperature.
Polyclonal rabbit antisera to P35 used at 1:10,000 was kindly provided by Paul Freisen
(University of Wisconsin, Madison). Polyclonal rabbit antisera to SERP2 used at 1:500
(424) and monoclonal mouse antisera to CrmA used at 1:2000 (236) have been
previously described. Following incubation with the primary antibody, membranes were
washed 3 times (5 minutes each) in wash buffer (PBS pH 7.4, 0.1% v/v Tween 20). The
membranes were then incubated with either 1:2500 goat anti-mouse IgG conjugated to

94
horseradish peroxidase or goat anti-rabbit IgG conjugated to horseradish peroxidase
(Southern Biotechnology Associates, Birmingham, AL) secondary antibodies in blocking
buffer for 1 hour at room temperature. This was followed by 5 washes (10 minutes each)
in wash buffer. Immunoreactive proteins were visualized by enhanced
chemiluminiscence using the ECL kit (Amersham) according to manufacturers
instructions.
Pulse Labeling
Confluent layers of LLC-PK1 cells (~8 x 105) in 12-well dishes were infected
with virus at MOI of 10. At 2 hours post infection (early time point) or 15 hours post
infection (late time point) medium was removed, and 300 pi of labeling medium (Cys',
*
Met', ICN Biomedicals, Irvine, CA) was added to each well. Cells were incubated for 30
min at 37C to deplete Cys and Met. After this 30 pi of 35S-translabel (ICN) was added
to each well, and labeling of proteins was allowed for 30 minutes. After labeling,
medium was removed, and cells were washed 2 x with radiolabel-ffee medium. Cells
were overlayed with 350 pi of radiolabel-free medium and incubated for various time
points (chase times). At appropriate intervals, cells were scrapped in the wells and
transferred to microfuge tubes. Samples collected (cells+supematants) were boiled with
SDS loading buffer for 15 minutes. One hundred microliter of each the collected sample
was resolved on 10% SDS-PAGE and subjected to autoradiography.

95
DNA Sequencing
Sequencing reactions were performed using 0.5 pg of plasmid DNA or 2pg of
genomic DNA, 4 pmols of primer, 3 pi of ABI Prism Dye Terminator Cycle Sequencing
Ready Reaction kit mix with AmpliTaq DNA polymerase (Perkin Elmer, Foster City,
CA), 2 pi of sequencing core reaction mix (University of Florida ICBR DNA Sequencing
Core Laboratory, Gainesville, FL) and water in a final reaction volume of 20 pi. Twenty-
five thermal cycles carried out according to manufacturers instructions (ABI Prism) in a
PTC-100 Thermal Controller (MJ Research Inc.). Each reaction was centrifuged in a
Millipore Ultrafree MC spin column (Millipore, Bedford, MA) to purify the DNA. The
resulting eluate was dried under vacuum, and the sequence was determined at the
University of Florida ICBR DNA Sequencing Core Laboratory, Gainesville, FL
following gel electrophoresis and analysis using an ABI 373 DNA sequencer.
Sequence Analysis and Database Search
Sequence searches were done using the BLAST search at the NCBI website
http://www.ncbi.nlm.nih.gov/BLAST/. Sequence comparisons and alignments were
performed using the Gap and Pileup programs of University of Wisconsin Genetics
Computing Group (GCG).
Recombinant Virus Construction
Transfection of Infected Cells using Liposomes
CV-1 cells were grown to 80% confluency in 6-well dishes in MEM with Earls
salts with 5% FBS. Medium was removed, and the cells were infected with virus (either

96
RPV or CPV) at 0.05 MOI in 500 pi serum-free medium (although the inclusion of
serum in the medium has no detrimental effect). Adsorption was allowed for 2 hours at
37C. Half an hour before adsorption was complete, plasmid DNA to be used to create
recombinant virus was diluted. Approximately 5 to 10 pg of plasmid DNA was diluted
to 50 pi in sterile distilled water. In a polystyrene tube, 30 pi of Lipofectin (or
Transfectace, Life Technologies) was diluted to 50 pi. The diluted DNA and diluted
lipofectin were mixed together in the polystyrene tube and allowed to form liposomes at
room temperature for 15 minutes. When virus adsorption was complete, the inoculum
was removed, and 1 ml of fresh serum-free medium was added to each well. The DNA
complex was then slowly added dropwise to the well with infected cells. Dishes were
incubated overnight at 37C. On the following day, an additional 1 ml of medium
containing 10% serum was added to each transfected well. After incubation for a further
period of 24 hours at 37C, the cells with medium were scrapped and collected in
microfuge tubes. Cells were stored at -80C until used. Prior to making serial dilutions
and plaquing virus, the cells were sonicated for 1 min.
Selection of Recombinant Virus in the Presence of Mycophenolic Acid
Mycophenolic acid (MPA) blocks purine metabolism and inhibits the formation
of vaccinia virus plaques in a number of cell lines (96). It has previously been shown that
inhibition by MPA can be overcome by expressing the E. coli guanine phosphoribosyl
transferase gene (Eco.gpt) in recombinant vaccinia virus in the presence of xanthine and
hypoxanthine in the medium. When constructing recombinant RPV or CPV expressing
Eco.gpt, the following procedure was followed.

97
To generate recombinant poxvirus expressing Eco.gpt, plasmids containing the
Eco.gpt gene cloned within the appropriate flanking regions where homologous
recombination is expected to occur were constructed. Cells infected with virus (either
RPV/CPV) were transformed with the Eco.gpt containing plasmid as described earlier.
Following transformation of cells, the virus mixture was serially diluted and inoculated
on CV-1 cells grown in 6-well dishes. Mycophenolic acid (25 pg/ml), xanthine (250
pg/ml) and hypoxanthine (15 pg/ml) were incorporated into the agar-medium just prior to
the addition of the overlay following the viral adsorption period. After 3 to 4 days of
growth, the cells were stained with neutral red, and plaques were visualized. One plaque
for each transformation reaction was picked with the help of a 500 pi micropipette and
re-suspended in 500 pi of PBS. This was termed the first plaque pick. The viral plaque
pick was sonicated, serially diluted and plaqued again on CV-1 cells and selected against
MPA. Following this first round of plaque purification, viral plaques were again picked
(second pick) and purified by another round of plaque purification in the presence of
MPA. Typically 5 such rounds of plaque purification were performed to produce pure
recombinant virus (RPV/CPV) expressing Eco.gpt.
When producing recombinant virus by transient dominant selection (TDS) as
described by Falkner and Moss (97), the Eco.gpt gene is cloned outside the flanking
regions that were used for homologous recombination. During homologous
recombination, the entire plasmid including the Eco.gpt gene is incorporated into the viral
genome under MPA selection and is maintained as an intermediate. Removal of MPA
selective pressure allows for concatemer resolution within the intermediate virus and the

98
emergence of the parental and recombinant virus. In addition to TDS, the use of a
screening marker such as P-galactosidase aids in identifying recombinant virus.
As described above, following transformation and plating of virus in the presence
of MPA, single viral plaques were selected during the first plaque pick. Subsequent
rounds of plaque purification were performed in the absence of MPA to allow for
concatemer resolution. At this stage of plaque purification, it would be useful to be able
to use P-galactosidase expression as an aid to select for plaques in the absence of MPA.
If the recombinant virus being constructed expresses P-galactosidase, then one would
screen for blue plaques in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-P-D-
galactopyranoside) (50 pi of 20 mg/ml X-Gal stock added to overlay of each well in 6-
well dish 12 hours prior to plaque picks). Alternatively, if the parental virus expressed P-
galactosidase, the recombinant virus was designed in such a way as to replace the P-
galactosidase marker and therefore one would select for non-P-galactosidase expressing
plaques or those that would be white instead of blue in the presence of X-Gal stain.
In the absence of any additional screening tool (such as p-galactosidase), multiple
MPA resistant plaques (typically 15) were picked in the first round of plaque purification.
Each plaque was purified independently. After 5 rounds of plaque purification, each
independently derived virus was grown in 60 mm dishes, and viral DNA was isolated.
The identification of recombinant virus was performed following PCR amplification of
the foreign gene from viral DNA. Typically the ratio of parental virus to recombinant
virus was consistently at approximately 9:1 respectively.

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Reverse Selection of Recombinant Virus using 6-Thioguanine
Expression of Eco.gpt can be used to generate recombinant virus in the presence
of 6-thioguanine (6TG), where selection is performed for virus lacking the Eco.gpt gene
(170). 6TG is a purine analog and is toxic to cells or virus expressing guanine
phosphoribosyl tranferase. Therefore cells such as STO cells used for reverse selection
lack the GPT gene. Briefly, STO cells were grown in the presence of 6TG in 6-well
dishes (0.2 mM for RPV and 0.4 mM for CPV). Following liposome-mediated
transformation of infected CV-1 cells as described earlier, the virus mixture was plated
on STO cells in the presence of 6TG. Viral plaques were stained 72 hours post infection
with neutral red. Typically four virus plaques were selected during the first viral pick and
purified five times by plaque purification in the presence of 6TG on STO cells. At this
stage, each independently derived virus clone was grown on CV-1 cells in 60 mm dishes,
and viral DNA was prepared. Identification of recombinant virus was performed
following PCR amplification of the foreign gene and confirming the lack of Eco.gpt
gene. A list of plasmids and viruses constructed in this study is listed in Tables 5 & 6.
Construction of Recombinant RPV Expressing CrmA
The upstream flanking sequence of the SPI-2 coding region was PCR amplified
with Vent polymerase (Amersham) from RPV genomic DNA using primers GM 17 (5-
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-31 containing
Xbal and Notl sites (underlined) and GM 18 (5-
TGCCTAGAATTCCATGGTAATCGATATTGGTCGTGT -3) containing Ncol and
EcoRI sites (underlined). The PCR product was cloned into the Xbal and EcoRI sites of

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pBluescript II KS(+) (Stratagene) resulting in pBS-RLF. The downstream flanking
sequence of the SPI-2 coding region was PCR amplified with from RPV genomic DNA
using primers GM 20 (5-
ATCG AT CGAATTCCCGGGC AT AT GCC ATTTTTTTT A A A A AA AAT AG A AAA A A
CATG-3) containing EcoRI, and Smal sites (underlined) and GM 22 (5-
TGCTACAAGCTTGATGAACACTGATTCCGCATC-3) containing a Hindlll site
(underlined). The right flank PCR product was cloned into the EcoRI and Hindlll sites of
pBS-RLF resulting in pBS-RCF.
The P7 5-gpt cassette that confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the EcoRI site of pBS-RCF resulting in
the shuttle vector pBS-Rgpt. RPVASPI-2::Eco.gpt was created using pBS-Rgpt and
wild type (wt) RPV by homologous recombination and selection of recombinant virus on
medium containing mycophenolic acid. The CrmA gene from pTMIHisCrmA (Peter
Turner unpublished) was re-cloned into the Ncol and Smal sites of pBS-RCF to generate
pRSG. The recombinant RPVASPI-2::CrmA virus was created by reverse selection on
STO cells using 6-thioguanine (170) using pRSG and RPVASPI-2::Eco.gpt.
Construction of Recombinant RPV Expressing P35
The P7 5-gpt cassette which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the Sail and Apal sites of pBS-RCF
resulting in the shuttle vector pBS-RgS/A.
The Pn-lacZ cassette which allows for blue-white screening with X-Gal (5-bromo-4-
chloro-3-indolyl-(3-D-galactopyranoside) was PCR amplified from pSCl 1 (61) with Vent

101
polymerase using primers FS 129 (5-
GTCAGATCCATGGTTGAATTCCGAGCTTGGCTG-3 ) containing the Ncol site and
FS 130 (5-AGTCAACGCCCGGGTACGCTCACAGAATTCCCG-3) containing the
Smal site. The lacZ PCR product was cloned into the Ncol and Smal sites of pBS-
RgS/A resulting in pRglacZ. Using pRglacZ the SPI-2 open reading frame in CPV was
replaced with lacZ by transient dominant selection (97) to make RPVASPI-2::lacZ.
A clone of the P35 gene from the baculovirus Autographa californica NPV was
provided by Lois Miller (University of Georgia, Athens). The plasmid pR35 was
constructed by inserting the P35 coding region between the Ncol and Smal sites of pBS-
RgS/A by recombinant PCR (421) such that the start codon of P35 exactly replaces the
ATG at the Ncol site. The primer pair used to generate the left flank was FS 1 (5-
GATCTCT AGAGCGGCCGCGGTTCGGT GGC A AACTTAC AT GGA A-3 ) containing
an Xbal site (underlined) and FS 90 (5-AGTAATCGATATTGGTCGTG-3 ). The
primer pair used to generate P35 was FS 88 (5-
CACGACCAATATCGATTACTATGTGTGTAATTTTTCCGG-3 ) (underlined portion
is complimentary to FS 90) and FS 74
(5 T AC GT C ACCCGGGTT ATTT AATT GTGTTT AAT ATTAC-3) containing a Smal
site at the 3end (underlined). Finally the left flank was linked to P35 using primers FS 1
and FS 74. The full length PCR product was cloned into the Xbal and Smal sites of pBS-
RgS/A resulting in pR35. The P35 recombinant virus RPVASPI-2::P35 was made using
pR35 and RPVASPI-2::lacZ by transient dominant selection and X-Gal staining of
recombinant viral plaques.

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Construction of Recombinant CPV Expressing SPI-2
The upstream flanking sequence of the CrmA coding region was PCR amplified
with Vent polymerase (Amersham) from CPV genomic DNA using primers GM 17 (5-
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-31 containing
Xbal and Notl sites (underlined) and GM 19 (5-
TCGATCGAATTCCATGGCAATCGATTTTGTTGT -3) containing Ncol and EcoRI
sites (underlined). The PCR product was cloned into the Xbal and EcoRI sites of
pBluescript II KS (+) (Stratagene) resulting in pBS-CLF. The downstream flanking
sequence of the CrmA coding region was PCR amplified with from CPV genomic DNA
using primers GM 21 (5- ATCGTAC
GAATTCCCGGGCATATGATCACATTCTTAATATTAGAA TATTAG-3) containing
EcoRI, and Smal sites (underlined) and GM 22(5-
TGCTACAAGCTIGATGAACACTGATTCCGCATC-3) containing a Hindlll site
(underlined). The right flank PCR product was cloned into the EcoRI and Hindlll sites of
pBS-CLF resulting in pBS-CCF.
The P7.5gpt cassette, which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the EcoRI site of pBS-CCF resulting in
the shuttle vector pBS-Cgpt. By transient dominant selection, CPVAcrmA::Eco.gpt was
created using pBS-Cgpt and wt CPV.
The SPI-2 gene from pTMlHisSPI-2 (Peter Turner unpublished) was re-cloned
into the Ncol and Smal sites of pBS-CCF to generate pCSG. The recombinant
CPVAcrmA:SPI-2 virus was created by reverse selection on STO cells using 6-
thioguanine (170) using pCSG and CPVAcrmA::Eco.gpt.

103
Construction of Recombinant CPV Expressing P35
The P7.5-gpt cassette which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the Sail and Apal sites of pBS-CCF
resulting in the shuttle vector pBS-CgS/A.
The Pn-lacZ cassette which allows for blue-white screening with X-Gal (5-bromo-4-
chloro-3-indolyl-P-D-galactopyranoside) was PCR amplified from pSCl 1 (61) with Vent
polymerase using primers FS 129 (5-
GTCAGATCCATGGTTGAATTCCGAGCTTGGCTG-3 ) containing the Ncol site and
FS 130 (5 AGTC AACGCCCGGGT ACGCT C AC AG AATTCCCG-3 ) containing the
Smal site. The lacZ PCR product was cloned into the Ncol and Smal sites of pBS-
CgS/A resulting in pCglacZ. Using pCglacZ the CrmA open reading frame in CPV was
replaced with lacZ by transient dominant selection (97) to make CPVAcrmA::lacZ.
The P35 gene from the baculovirus Autographa californica NPV was provided by
Lois Miller (University of Georgia, Athens). The plasmid pC35 was constructed by
inserting the P35 coding region between the Ncol and Smal sites of pBS-CgS/A by
recombinant PCR (421) such that the start codon of P35 exactly replaces the ATG at the
Ncol site. The primer pair used to generate the left flank was FS 1 (5-
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-3 ) containing
an Xbal site (underlined) and FS 91 (5 -GGCAATCGATTTTGTTG-3 ). The primer pair
used to generate P35 was FS 89 (5-
CAACAAAATCGATTGCCATGTGTGTAATTTTTCCGG-3 1 (underlined portion is
complimentary to FS 91) and FS 74(5-
T ACGT C ACCCGGGTT ATTT AATT GT GTTTA AT ATT AC-3 ) containing a Smal site

104
at the 3end (underlined). Finally the left flank was linked to P35 using primers FS 1 and
FS 74. The full length PCR product was cloned into the Xbal and Smal sites of pBS-
CgS/A resulting in pC35. The P35 recombinant virus CPVAcrmA::P35 was made using
pC35 and CPVAcrmA::lacZ by transient dominant selection and X-Gal staining of
recombinant viral plaques.
Construction of Recombinant CPV Expressing SERP2
The SERP2 gene was re-cloned from pTMlSERP2 (Peter Turner unpublished)
into the Ncol and Smal sites of pBS-CgS/A resulting in pCSERP2. Using pCSERP2 and
CPVAcrmA::lacZ, recombinant virus CPVAcrmA::SERP2 was made by transient
dominant selection and X-Gal staining of viral plaques.
Site Directed Mutagenesis of the CrmA Gene
The PI aspartic acid residue at position 303 within CrmA was mutated to alanine
using the Altered Sites mutagenesis system (Promega) following the manufacturers
instructions. Briefly, the CrmA coding region was cloned into the EcoRI and Hindlll
sites of pAlterEX-1. To generate the D303A mutation, a 5-phosphorylated
oligonucleotide, FS 275 (5-p-
TGT GCGCTGGT GGC AGCATGCGC ATC AAC AGTTAC A)
(mismatches underlined) was used. The mismatch generated an SphI site that was used
for screening purposes. The resulting plasmid construct, pAlterEx-lD303ACrmA,was
verified for the mutation by sequence analysis.

105
Construction of CPVCrmA D303A
The D303A mutant CrmA coding region was PCR amplified using FS 307 (5-
TACGTCCATGGATATCTTCAGGGAAA-3) containing an Ncol site (underlined) and
FS 308 (5-CAGCTACCCGGGTTATAATTAGTTGTTGGAGAGC-3) containing a
Smal site (underlined). The PCR product was cloned into the Ncol and Smal sites of
pBS-CgS/A resulting in pBS-D303ACgS/A. Using CPVAcrmA::lacZ and pBS-
D303ACgS/A, CPVCrmA D303A was made by transient dominant selection and X-Gal
staining of viral plaques. The viral construct was verified by sequence analysis and for
expression of mutant CrmA by immunoblot.
Construction of CPVAIL-1 pR Virus
The CPV IL-1P receptor is designated as ORF B14R (based on the vaccinia virus
sequence) and corresponds to base pairs 194007-194987 in the CPV genome. The design
of the knockout plasmid was such that the P7 5-gpt cassette that confers resistance to
mycophenolic acid (96) was cloned between the first 260 base pairs of CPV IL-1 p
receptor (left flanking sequence) and the last 223 base pairs of the viral receptor (right
flanking sequence). The left flanking sequence was PCR amplified with Vent
polymerase (Amersham) from CPV genomic DNA using primers FS 403 (5-
AGTC AT GAATTC AGT AT ACC ACCT GTT AT-3 ) containing EcoRI site (underlined)
and FS 413 (5-ACTCCCTGCAGTCATTAATCATTATCCGCTCCTCG-3) containing
PstI site (underlined). The left flanking PCR product was cloned into the EcoRI and PstI
sites of pBluescript II KS(+) (Stratagene) resulting in pBS-CPVIL-lR-LF.

106
The P7 5-gpt cassette which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and PCR amplified amplified with Vent polymerase
(Amersham) using primers FS 414 (5-
TCAGACTGCAGCCCGGTAGTTGCGATATAC-3) containing the PstI site
(underlined) and FS 415 (5 -TGACGCCCGGG AG ATTAGCG ACCGGAG ATT-3 )
containing the Smal site (underlined). The Eco.gpt PCR product was cloned into the PstI
and Smal sites of pBS-CPVIL-lR-LF to generate pBS-CPVIL-lR-LF-MPA.
The right flanking sequence was PCR amplified from CPV genomic DNA using
primers FS 416 (5 -AGCTGCCCGGGCGTTGAGACCTCCCACAACG-3 ) containing
the Smal site (underlined) and FS 408 (5-
CTGC ACGGATCCC AATC ACGTTATACTAATAGTAAC-3 ) containing the BamHI
site (underlined). The right flanking PCR product was cloned into the Smal and BamHI
sites of pBS-CPVIL-lR-LF-MPA resulting in pBS-CPVIL-IRKO.
By mycophenolic acid selection, CPV AIL-1 pR::Eco.gpt virus was created using pBS-
CPVIL-1RKO and wt CPV.
Apoptosis Assays
DAPI Staining of Infected Cells
Cells undergoing apoptosis were visualized by staining DNA within cells. LLC-
PK1 cells were grown to 80% confluency in eight-well chamber slides (LabTek,
Campbell CA) and infected at an MOI of 10. After 2 hour adsorption, the inoculum was
removed, and 200 pi of Medium 199 was added. The cells were further incubated for 14
hours and then washed with 300 pi of PBS. Cells were fixed in 200 pi of PBS containing

107
Table 5. List of plasmids constructed
Plasmid
Properties
1. pBS-RLF
SPI-2 left flank in pBlueScript Xbal and EcoRI
2. pBS-RCF
SPI-2 right flank in pBS-RLF EcoRI and Hindlll
3. pBS-Rgpt
Eco.gpt in pBS-RCF EcoRI
4. pRSG
CrmA in pBS-RCF Ncol and Smal
5. pBS-RgS/A
Eco.gpt in pBS-RCF Sail and Apal
6. pRglacZ
LacZ in pBS-RgS/A Ncol and Smal
7. pR35
P35 in pBS-RgS/A by recombinant PCR
8. pBS-CLF
CrmA left flank in pBlueScript Xbal and EcoRI
9. pBS-CCF
CrmA right flank in pBS-CLF EcoRI and Hindlll
10.pBS-Cgpt
Eco.gpt in pBS-CCF EcoRI
ll.pCSG
SPI-2 in pBS-CCF Ncol and Smal
12.pBS-CgS/A
Eco.gpt in pBS-CCF Sail and Apal
13.pCglacZ
LacZ in pBS-CgS/A Ncol and Smal
14.pC35
P35 in pBS-CgS/A by recombinant PCR
15.pCSERP2
SERP2 in pBS-CgS/A Ncol and Smal
1 .pAlterEX-1 CrmA
CrmA in pAlterEX-1 EcoRI and Hindlll
17.pAlterEx-1D303 ACrmA
CrmA D303A generated by mutagenesis
18.pBS-D303ACgS/A
CrmA D303A in pBS-CgS/A
19.pBS-CPVIL-1R-LF
CPVvIL-ipR left flank in pBluescript EcoRI and PstI
20.pBS-CPVIL-1R-LF-MPA
Eco.gpt in pBS-CPVIL-1 R-LF PstI and Smal
21 .pBS-CPVIL-1RKO
CPVvIL-lpR right flank in pBS-CPVIL-1 R-LF-MPA

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Table 6. List of constructed recombinant viruses
Recombinant Viruses Constructed
Virus and plasmids used
1. RPVASPI-2::Eco.gpt
RPV and pBS-Rgpt
2. RPVASPI-2::CrmA
RPVASPI-2::Eco.gpt and pRSG
3. RPVASPI-2::lacZ
RPV and pRglacZ
4. RPVASPI-2::P35
RPVASPI-2::lacZ and pR35
5. CPVAcrmA::Eco.gpt
CPV and pBS-Cgpt
6. CPVAcrmA:SPI-2
CPVAcrmA: :Eco.gpt and pCSG
7. CPVAcrmA::lacZ
CPV and pCglacZ
8. CPVAcrmA::P35
CPVAcrmA::lacZ and pC35
9. CPVAcrmA::SERP2
CPV AcrmA: :lacZ and pCSERP2
lO.CPVCrmA D303A
CPV AcrmA: :lacZ and pBS-D303ACgS/A
ll.CPVAIL-lpR::Eco.gpt
CPV and pBS-CPVIL-IRKO

109
3.5% formaldehyde for 20 minutes at room temperature followed by 200 pi of cold
methanol for 10 minutes. Following two rinses with 300 pi of PBS, the cells were
stained with 100 pi of PBS containing 0.5 pg/ml of DAPI (4, 6-diamidino-2-
phenylindole) for 30 minutes at room temperature. This was followed by three final
rinses in 300 pi of PBS. The DNA was visualized using a fluorescence microscope
equipped with a DAPI filter. Cells were photographed with Fuji 400ASA film.
Preparation of Infected Cell Extracts for Caspase Activity Assay
Confluent monolayers of LLC-PK1 cells (~2 x 106) grown in 6-well
dishes were either mock infected or with the relevant virus at an MOI of 10 in 500 pi of
Medium 199 without serum. Following 2 hours of adsorption at 37C, the inoculum was
removed, and 950 pi of fresh Medium 199 (without serum) was added. After 14 hours of
incubation at 37C, the cells were harvested by scraping into medium, and to this was
added 100 pi of extract buffer (100 mM HEPES, pH 7.5; 20 mM EDTA; 1% CHAPS; 10
mM dithiothreitol [DTT]), freeze-thawing three times and clarifying by centrifugation at
12,000 x g for 5 minutes. Protein concentrations were determined by a modified
Bradford assay using the Coomasie kit (Pierce).
Ac-DEVD-AMC Cleavage Assay for Caspase Activity
Terminal caspase activity was determined as increase in fluorescence with time of
the substrate Ac-DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl coumarin)]
(Bachem) used at 10 pM with 25 pg of extract in 200 pi of caspase buffer (100 mM
HEPES, pH 7.5; 10% sucrose; 0.1% CHAPS; 10 mM DTT). Cleavage of the substrate

110
was monitored using a Tecan SpectraFluor microplate reader to detect free amino methyl
coumarin, with an excitation at 380 nm and an emission at 460 nm. Values were
expressed as rates of fluorescence increase per second.
In Vivo Infection Assays using CAMs
Preparation of Chicken Chorioallantoic Membranes for Infections
Embryonated chicken eggs were obtained from SPAFAS, Inc (Roanoke, IL) and
incubated at 38.5C with 50% humidity for 11 days. The position of the air sac, blood
vessels, yolk sac and growing chick embryo was noted by candling the egg against a
source of light from a table lamp. The region of shell at the air sac was swabbed with
95% alcohol. The first incision (-0.5-1 mm) was made in the shell at the air sac region
with the tip of a modified file. The tip of the file was dipped in 95% alcohol and flamed
prior to making the incision. Care was taken not to puncture the air sac. A region (-1 cm
in diameter) about the middle of the egg (chosen based on the absence of yolk sac and
large blood vessels) was swabbed with Prepodyne solution (1% iodine) (WestAgro,
Kansas, MO) and air dried. A smaller region (-0.5 cm in diameter) was swabbed with
95% alcohol inside the area cleaned by Prepodyne. Swabbing with alcohol was repeated
approximately five times. The egg was positioned with the freshly swabbed region at the
apical surface. Very gently, a second incision (0.5-1 mm) was made in the shell at the
center of the swabbed region. Aseptic precautions were taken by flaming the tip of the
file dipped in 95% alcohol prior to making the incision. Care was taken not to puncture
the chorioallantoic membrane just beneath the shell and shell membrane. The air in the
air sac was gently removed using a rubber bulb placed at the incision made over the air

Ill
sac. Due to negative pressure created by the deflated air sac, the chorioallantoic
membrane detached from the shell and was suspended below the second incision. Eggs
with dropped CAMs were always oriented with the CAM forming the apical surface.
Following aseptic introduction of virus onto the CAM, the incisions were sealed with
adhesive tape.
Infecting and Harvesting CAMs
The dropped CAMs were inoculated aseptically with 10 to 100 pock-forming
units (PFU) of virus in a lcc syringe tipped with 27.5 G needle. The inoculated eggs were
further incubated at 38.5C for 72 hours on a tabletop incubator without shaking. At the
end of the incubation period, the CAMs were harvested from the eggs. Briefly, the egg
was held over a disposal container, and the region of shell directly on the opposite side of
the CAM (underside of the positioned egg) was cracked open gently. Using a large
scissor, the shell was cut open slowly as the contents were allowed to collect in the
disposal container. The chick embryo was destroyed in the process to prevent the
embryo from pulling apart the entire CAM. The CAM was carefully detached from the
empty eggshell with a pair of forceps and placed in a petri dish containing phosphate
buffered saline (PBS) (pH 7.2). The harvested CAMs were washed twice in PBS and
scanned on a Microtek Scanmakerlll flatbed scanner at 600dpi. Individual pocks were
excised and stored at -80C. Alternatively, membranes inoculated with 500 PFU were
harvested at 48 hours post infection to yield confluently infected membranes, which were
stored at -80C till processed for extracts.

112
Measurement of Reactive Oxygen Intermediates
The CAMs from eggs were harvested and washed twice with PBS. The
membranes were then incubated in 0.1% nitroblue tetrazolium (NBT) (Sigma Chemical
Co.) in PBS at 37C for 1 hour. The NBT solution was removed, membranes were
washed once with PBS and then scanned at 600 dpi. The NBT is reduced to dark blue
insoluble formazan within pocks indicating the presence of reactive oxygen
intermediates.
MTT Reduction Assay
This assay quantitates the amount of reactive oxygen intermediates that may be
present within CAMs. Confluently infected membranes harvested at 48 hours post
infection were excised. Approximately 20mg pieces of membranes were incubated in
200pl of PBS containing 5 mg/ml 3,(4,5-dimethylthiazoI-2yl)-2,5-diphenyltetrazolium
(MTT) for 30 minutes at 37C. The MTT solution was removed, and the reduced
formazan was extracted by grinding the membranes with a microfuge pestle in 200pl of
dimethyl sulfoxide (DMSO) (Sigma). The dissolved formazan was clarified in microfuge
tubes by centrifugation at 12,000 x g for 2 minutes. The absorbance of the resulting
solution was read at 550nm. The absorbance values were expressed as OD/mg of tissue.
Virus Infectivity Assay
Individual virus pocks were isolated and excised from infected membranes. The
pocks were ground using a microfuge pestle in 500pl of PBS, and the cells lysed by three

113
cycles of freeze-thawing at -70 and 25C. The crude lysates were plaqued on CV-lcells
and viral titers were expressed as PFU/pock.
Terminal Caspase Activity Assay on Extracts from Infected CAMs
Individual pocks were isolated from infected membranes. Extracts of pocks were
made by grinding pocks using a microfuge pestle in lOOpl of extract buffer (10 mM
HEPES, pH 7.5; 2 mM EDTA; 0.1% CHAPS; 1 mM dithiothreitol [DTT]), freeze
thawing three times and clarifying by centrifugation at 12,000 x g for 5 minutes. The
protein concentrations in collected supernatants were determined by Bradford assay in a
microplate reader. Terminal caspase activity was determined as increase in fluorescence
with time of the substrate Ac-DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl
coumarin)] (Bachem) used at 10 pM with 25 pg of pock extract in 200 pi of caspase
buffer (100 mM HEPES, pH 7.5; 10% sucrose; 0.1% CHAPS; 10 mM DTT). Cleavage
of the substrate was monitored using a Tecan SpectraFluor microplate reader to detect
free amino methyl coumarin, with an excitation at 380 nm and an emission at 460 nm.
Values were expressed as rates of fluorescence increase per second.
Alternatively, CEF cells were grown to 80% confluency in 12 well dishes
(Costar). The cells were infected with virus at a multiplicity (MOI) of 10. After 2 hours
of absorption (9 hours for CEF cells) the inoculum was removed and fresh Medium 199
without serum was added. The cells and supernatants were harvested at different time
points and lysed in extract buffer. The extracts were processed for terminal caspase
activity using the substrate Ac-DEVD-AMC.

114
Construction of Plasmids Containing Chicken ProIL-ip or ProIL-18
Since caspase-1 processes proIL-ip and proIL-18 to the mature cytokines, we
wanted to test CAM extracts for the ability to process radiolabeled chicken cytokine
precursors transcribed and translated in vitro. Plasmid cDNA clones containing the
coding sequence of chicken proIL-1 p or proIL-18 were kindly provided by Peter Staeheli
(University of Freidburg, Germany). The chicken proIL-ip gene was PCR amplified
from the original plasmid cDNA clone using Vent polymerase (New England Biolabs)
with primers FS 401 (5-ACGTAGCCATGGCGTTCGTTCCCGACCTG-3) at the 5
end of the open reading frame containing an added Ncol site (underlined) and FS 402 (5-
GCATTCGGATCCTCAGCGCCCACTTAGCT-3) at the 3 engineered to contain a
BamHI site. Similarly the chicken IL-18 gene was amplified with primers FS 409 (5-
GCTTACTCATGAGCTGTGAAGAGA-3) at the 5 end of the open reading frame
containing an added BspHI site (underlined) and FS 410 (5-
ACTTCGGGATCCTGATCATAGGTTGTGCCT-3) at the 3 engineered to contain a
BamHI site. The PCR products were cloned into the Ncol and BamHI sites of pTMl
(272) resulting in pTMlchIL-ip containing the chicken proIL-ip coding sequence and
pTMlchIL-18 containing the chicken proIL-18 sequence. The plasmid constructs were
oriented such that the open reading frames could be expressed from the T7 promoter of
with each plasmid. 35S-labelled chicken proIL-ip and proIL-18 were synthesized using
the T7 Quick Coupled Transcription/Translation System (TNT) (Promega Corporation)
and Trans 35S-Label (ICN) as the source of [35S] methionine according to the
manufacturers instructions.

115
Plasmid Containing Mouse ProIL-ip
A cDNA clone of mouse proIL-ip was kindly provided by Rudy Beyaert,
University of Ghent, Belgium. The orientation of the construct pGEMl 1-ILlbeta
containing mouse proIL-ip was such that transcription was possible using the SP6
promoter within the plasmid. In vitro radiolabeled proteins were synthesized using the
SP6 TNT system (Promega) according to manufacturers instructions.
Quick Coupled Transcription/Translation System
The Quick Coupled Transcription/Translation System (TNT) (Promega) is a
single-tube reaction for in vitro transcription and translation from plasmid constructs
containing the appropriate promoters for RNA polymerases such as SP6 and T7. The
TNT system contains a single mixture of RNA polymerase, nucleotides, salts, RNAse
inhibitors and rabbit reticulocyte lysate to form a master mix. Briefly, the reactions were
performed as follows. Typically 0.5-1 pg of plasmid DNA was added to 20 pi of
aliquoted TNT master mix. To this mixture was added [35S] methionine (10 pCi) (ICN),
and the volume brought up to 25 pi. The TNT reaction was incubated at 30C for 60
minutes. After the reaction is complete, the tubes were placed on ice if used immediately
or frozen at -20C until used. Typically for constructs in pTMl background, 1 to 2 pi of
TNT reaction was sufficient to be visualized following SDS-PAGE and autoradiography.
In Vitro Cleavage Assay for Processing ProIL-ip and ProIL-18
Cytokine processing activity was determined as follows. Two microliter of 35S-
labelled proIL-ip or proIL-18 from 25 pi TNT reaction was incubated with 1 unit of

116
recombinant human caspase-1 or 15U of recombinant human caspase-3, (kindly provided
by Nancy Thombery, Merck Research Laboratories, Rahway, N.J.) in 100 pi (final
volume) caspase buffer (100 mM HEPES, pH 7.5; 10% sucrose; 0.1% CHAPS; 10 mM
DTT) for 4 hours at 37C. The proteins were resolved on 10 % sodium dodecyl sulfate-
polyacrylamide gels (SDS-PAGE), the radioactive signal was enhanced with Amplify
(Amersham) and the proteins visualized by autoradiography.
Alternatively, confluently infected membranes (2-3 membranes for each
infection), harvested at 48 hours post infection, were homogenized in extract buffer (final
volume 800 pi), and protein concentrations were determined by Bradford assay. 200 pg
of CAM extracts were incubated with 2 pi of S-labeled proIL-1 P or proIL-18 derived
from TNT reactions in 100 pi (final volume) Caspase buffer. The protein mixtures were
incubated for 4 hours at 37C. Following incubation the proteins were subjected to SDS-
PAGE and the gels enhanced with Amplify (Amersham). Radiolabeled proteins were
visualized by autoradiography.
When using peptide inhibitors for caspases, the CAM extracts or purified human
caspase was pre-incubated with either 10 nM of Ac-DEVD-CHO [acetyl-Asp-Glu-Val-
Asp-aldehyde] (Bachem) or 100 nM of Ac-WEHD-AMC [acetyl-Trp-Glu-His-Asp-
aldehyde] (Bachem) or 200 pi of Z-VAD-FMK [benzyloxycarbonyl-Val-Ala-Asp-(OMe)
fluromethyl ketone] (Bachem) at 37C for 2 hours prior to the addition of radiolabeled
proIL-ip or proIL-18. Caspase activity was determined by the position of the cleaved,
mature cytokine products that are smaller than the radiolabeled precursor and therefore
migrate faster on SDS-PAGE.

CHAPTER 3
RESULTS
Equivalency of SPI-2 and CrmA
The question of whether or not RPV SPI-2 and CPV CrmA are functionally
equivalent in vivo can be answered by interchanging the genes between the two viruses or
by genetic complementation experiments. LLC-PK1 cells infected with CPV fail to
undergo apoptotic induction, whereas CPVAcrmA infections of these cells induce
apoptosis indicating that CrmA functions to inhibit apoptotic induction during virus
infection (236). RPV or RPVASPI-2 infections of LLC-PK1 cells induce apoptosis.
Mixed infections of LLC-PK1 cells by RPVASPI-2 or RPV in the presence of CPV (but
not CPVAcrmA) failed to induce apoptosis. This proved that CrmA encoded by CPV
could inhibit the RPV induced apoptosis of pig cells. However, mixed infections of pig
cells with CPVAcrmA and RPV, induced apoptosis, indicating that SPI-2 encoded by
RPV was unable to prevent apoptotic induction and substitute for CrmA in the mixed
infection. Therefore by genetic complementation experiments it has been shown that
while CrmA can substitute for SPI-2, the converse was not true. Since we are dealing
with two different viral genomes, it would be more accurate to address the differences
between the two molecules if the genes were swapped between the viruses. Thus we
wanted to assess the functions of SPI-2 and CrmA within different viral contexts to
determine equivalency.
117

118
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Recombinant Virus Construction
The strategy we choose to employ involved replacing the entire coding region of
CrmA with that of SPI-2, and vice-versa. Separate plasmid vectors containing the left
and right flanks of either SPI-2 or CrmA interspersed by E.coli gpt were constructed as
shown in Figures 8 and 9. Initially, knockout viruses were constructed in which the SPI-
2 or CrmA genes were deleted and replaced by E. coli gpt in RPV and CPV, respectively,
by homologous recombination and MPA selection to generate RPVASPI-2::Eco.gpt and
CPVAcrmA::Eco.gpt (see Recombinant Virus Construction in Materials and Methods).
Plasmid vectors (pRSG) containing CrmA gene flanked by left and right SPI-2 flanking
regions were also created. Similar constructs (pCSG) were made for the SPI-2 gene.
Using RPVASPI-2::Eco.gpt and pRSG, recombinant RPV virus expressing CrmA
(RPVASPI-2::crmA) was constructed by reverse selection in STO cells (170). Similarly
CPVAcmA::Eco.gpt virus and pCSG were used to construct CPV expressing SPI-2
(CPVAcrmA::SPI-2) by reverse selection in STO cells.
The two viruses were sequenced to confirm the foreign gene inserts and tested for
the expression of SPI-2/CrmA by immunoblot analysis as seen in Figures 10 and 11. The
controls showed that SPI-2 expressed in RPV migrates at 38 kDa (Fig. 10). In addition
another immunodominant band migrating at approximately 36 kDa began to appear at 10
hours post infection and persisted throughout the RPV infection. This could possibly
represent a degraded/cleaved form of SPI-2 in RPV, although the levels to uncleaved
SPI-2 in RPV infections did not diminish at all. CrmA expressed in the context of RPV
migrated at 38 kDa as expected and was stable throughout the virus infection up to 18

119
hours post infection (Fig. 10). As in the case of SPI-2, there were also present
cleaved/degraded forms of CrmA produced during RPV infections, although to a much
lesser extent.
CrmA expressed by wt CPV migrated as expected (Fig. 11). SPI-2 expressed
from CPV in place of CrmA was stable through out virus infection as the converse
construct seen in (Fig.l 1). SPI-2 expressed in CPV did not seem to be degraded/cleaved
as in wt RPV infections (compare Fig. 10 and 11). It is apparent that SPI-2 and CrmA
have been successfully swapped or interchanged between RPV and CPV, respectively,
and that the mutant viruses express the recombinant proteins.
The replacement of SPI-2 with CrmA in RPV and the converse construct in CPV
allowed us to examine the effect of the activity of these genes within different viral
contexts during infections of LLC-PK1 cells.
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Prevent Apoptosis in Pig Cells
In order to test the equivalency of the SPI-2 and CrmA we swapped the genes
in the parent viruses and used the recombinant viruses to infect swine cells. Infected cells
were stained with DAPI and viewed microscopically as shown in Figures 12 and 13.
Consistent with previous reports, wt CPV-infected cells do not appear apoptotic whereas
CPVAcrmA::lacZ induced the morphological changes seen in apoptotic cells such as
densely staining apoptotic bodies (Fig. 13) (236). SPI-2 replacement of CrmA in CPV
behaved like wt CPV and did not induce apoptotic changes in swine cells suggesting that
SPI-2 could functionally substitute for CrmA function in CPV (Fig. 13). Both RPV and
RPVASPI-2 infections induced the formation of apoptotic cells in agreement with

120
A.
SPI-2
178,373 179,407
RPV Genome
197,732 bp
GM 17
SPI-2 ORF 20
B
GM 18
GM 22
B.
Xbal
LacZ
Hindlll
Xbal ,c Hindlll
SPI-2 Uft Hank (1005)
Figure 8. Construction of recombinant RPV. (A) The location of SPI-2 within the
RPV chromosome is indicated. Left and Right flanks (hatched boxes) of the SPI-2
coding region were amplified using the primers indicated. (B) Illustration of the shuttle
vector used to replace SPI-2 in RPV with either lacZ, or P35 genes. The plasmid pBS-
RgS/A contains the E.coli gpt gene outside the SPI-2 flanks (hatched boxes). The various
genes were cloned into pBS-RgS/A, which was then used to generate recombinant RPVs
by transient dominant selection (97). Restriction sites used for cloning are indicated. See
Materials and Methods (Recombinant Virus Construction) for each individual virus
construct.

121
A.
CPV Genome
1
CrmA
192,309 193,334
\/
224,501 bp
GM 17
CrmA ORF
GM 21
GM 19
GM 22
B.
Xbal
LacZ
Hindi II
Xbal
P35
Ncol
SERP2
Smal
Ncol
CrmA D303A
Smal
CrraA Left Rank
Apal (2708)
Figure 9. Construction of recombinant CPV. (A) The location of the CrmA gene
within the virus chromosome is indicated. The flanks upstream and down stream
(hatched boxes) of the CrmA coding region were amplified using the primers indicated.
(B) Illustration of the shuttle vector used to replace CrmA in CPV with either lacZ, P35,
SERP2 or CrmA D303A. The various genes were cloned into pBS-CgS/A containing the
E.coli gpt gene outside the CrmA flanks (hatched boxes), which were then used to
generate recombinant CPVs by transient dominant selection (97). Restriction sites used
for cloning are indicated. See Materials and Methods (Recombinant Virus Construction)
for each individual virus construct.

122
Wt RPV
hrp.i 2 6 10 12 14 16 18
49 kDa -
SPI-2
36 kDa -
1 2 3 4 5 6 7
RPVASPI-2::crmA
hrp.i 2 6 10 12 14 16 18 Mock
49 kDa -
CrmA
36 kDa -
1 2 3 4 5 6 7 8
Figure 10. Expression of SPI-2 and CrmA during RPV infections. Extracts were
made from CV-1 cells infected at an MOI of 10 harvested at various times post infection,
subjected to SDS-PAGE and immunoblotted with monoclonal antisera to (A) SPI-2
expressed in wt RPV and (B) CrmA expressed in RPVASPI-2::crmA. Arrows indicate
the position of migrating SPI-2/crmA proteins at 38 kDa. Immunoblots indicate that the
recombinant proteins are being expressed during virus infection. A lower-molecular
weight protein is also seen at late times in both infections and may represent a cross
reacting band or a degradation product of SPI-2/CrmA.

123
A.
Wt CPV
hr p.i 2 6 8 10 12 14 16 18
CrmA
36 kDa -
1 2 3 4 5 6 7 8
B.
hr p.i
SPI-2
36 kDa -
1 2 345 6789
CPVAcrmA::SPI-2
2 6 8 10 12 14 16 18 Mock
Figure 11. Expression of CrmA and SPI-2 during CPV infections. Extracts were
made from CV-1 cells infected at an MOI of 10 harvested at various times post infection,
subjected to SDS-PAGE and immunoblotted with monoclonal antisera to (A) CrmA
expressed in wt CPV and (B) SPI-2 expressed in CPVAcrmA::SPI-2. Arrows indicate the
position of migrating CrmA/SPI-2 proteins at 38 kDa. Immunoblots indicate that the
recombinant proteins are being expressed from during virus infection.

124
previous reports (Fig. 12) (236). However CrmA replacements of SPI-2 in RPV, looked
similar to wt CPV infections suggesting that SPI-2 and CrmA did not function in an
identical fashion within the context of RPV infections (compare Fig. 12 and 13). These
observations strengthened our hypothesis that despite their high degrees of similarity,
SPI-2 and CrmA were not functionally equivalent. In order to confirm our results seen
morphologically by DAPI stains, we also analyzed virus-infected cell extracts for caspase
induction that would demonstrate the biochemical changes seen in apoptotic cells (see
below).
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Inhibit Caspase Activation
The induction of apoptosis is characterized biochemically (most of the time) with
the activation of caspases. To assess caspase activity in virus-infected cells, extracts
were made at different times post infection. Cell extracts were assayed for the ability to
cleave Ac-DEVD-AMC, a fluorogenic peptide that is specific for terminal caspase-3-
like enzymes (Figure 14 and 15). Cleavage of Ac-DEVD-AMC is indicative of caspase
activity typically present in apoptotic cells. Extracts were made from wt and recombinant
RPV infected cells and assayed for caspase activity by incubating a portion of the extract
with the fluorogenic peptide Ac-DEVD-AMC (Fig. 14). Peptide cleavage was
determined as a function of time and expressed as rate of cleavage per second. RPV
containing functional SPI-2 gene induced caspase activity (Fig. 14). Deletion of SPI-2
from RPV induced Ac-DEVD-AMC cleaving activity after 6 hours post infection with a
maximum rate at 12 hours post infection as shown in Figure 14. We also observed that
the rate of Ac-DEVD-AMC cleavage increased with time and stabilized at late times post

125
A. Mock B. RPV
C. RPVASPI-2::lacZ D. RPVASPI-2::crmA
E. RPVASPI-2::P35
Figure 12. Morphological characteristics of LLC-PK1 cells infected with RPV
derivatives. Pig kidney cells were infected at MOI 10 and stained with DAPI at 16 hours
post infection. Panels indicate the different viruses used for infection (A) Mock, (B)
RPV, (C) RPVASPI-2::lacZ, (D) RPVASPI-2::crmA and (E) RPVASPI-2::P35.
Examples of apoptotic bodies, characteristic of cells undergoing apoptosis are indicated
by arrowheads.

126
A. Mock
B. CPV
D. CPVAcrmA::SPI-2
, ^ ****
¡ft 9
**
* m
E. CPVAcrmA::P35
mm
. 5*
A 9
m
j
Figure 13. Morphological characteristics of LLC-PK1 cells infected with CPV
derivatives. Pig kidney cells were infected at MOI 10 and stained with DAPI at 16 hours
post infection. Panels indicate the different viruses used for infection (A) Mock, (B)
CPV, (C) CPVAcrmA::lacZ, (D) CPVAcrmA::SPI-2 and (E) CPVAcrmA::P35.
Examples of apoptotic bodies, characteristic of cells undergoing apoptosis are indicated
by arrowheads.

127
infection (12 to 18 hours). It is apparent that SPI-2 in RPV infections is incapable of
completely blocking caspase activation. These results show that SPI-2 does function to
reduce caspase induction although SPI-2 is unable to completely block caspase induction.
More interestingly, in extracts from RPVASPI-2::crmA infections, (CrmA replacement of
SPI-2 in RPV) induction of caspase activity was completely blocked (Fig. 14). These
results suggest that CrmA functions more efficiently than SPI-2 in the context of RPV
and that unlike SPI-2, CrmA was able to completely block the activation of terminal
caspases during RPV infections of LLC-PK1 cells.
Wild type CPV infections of LLC-PK1 cells do not induce caspase activity
(Fig. 15). However CPVAcrmA::lacZ infections (like RPVASPI-2::lacZ and to a lesser
extent RPV), induce caspases as early as 8 hours post infection and reach maximum rates
at 12 hours post infection similar to those seen with RPVASPI-2. SPI-2 replacements of
CrmA in CPV (CPVAcrmA::SPI-2) function within the context of CPV to completely
block caspase induction just like wt CPV infections (Fig. 15). Therefore in CPV
infections of pig kidney cells, SPI-2 and CrmA function in a similar fashion with the
dissimilarities between the two proteins only apparent when expressed from within RPV.
SPI-2 and CrmA are similar with respect to kinetics of expression and inhibitory
profiles based on in vitro studies (236). Our results validate previous observations seen
for CrmA and SPI-2 in genetic complementation experiments (236). Thus, despite the
similarities observed between SPI-2 and CrmA in vitro, our results imply that RPV SPI-2
and CPV CrmA are not completely functionally equivalent in vivo during infections of
swine cells.

128
Figure 14. Biochemical changes in LLC-PK1 cells infected by RPV derivatives. Pig
kidney cells were infected at MOI10 and harvested at various times post infection. Cell
extracts were made and incubated with terminal caspase substrate Ac-DEVD-AMC, a
fluorogenic peptide. Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Ac-
DEVD-AMC cleavage rates were determined at each harvested time point and are
expressed arbitrarily as fluorescence signal units (FSU) per second. Legends
corresponding to the different viruses tested are indicated.

129
hr p.i.
Figure 15. Biochemical changes in LLC-PK1 cells infected by CPV derivatives. Pig
kidney cells were infected at MOI 10 and harvested at various times post infection. Cell
extracts were made and incubated with terminal caspase substrate Ac-DEVD-AMC, a
fluorogenic peptide. Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Ac-
DEVD-AMC cleavage rates were determined at each harvested time point and are
expressed arbitrarily as fluorescence signal units (FSU) per second. Legends
corresponding to the different viruses tested are indicated.

130
Recombinant RPV and CPV Viruses Expressing P35
Initially we chose to study the function of both SPI-2 and CrmA in regulating
caspase induction in vivo by respective RPV and CPV viruses. If SPI-2/CrmA function
to regulate apoptosis solely by interacting with caspases, then replacing these genes with
other potent inhibitors of caspases should produce similar effects during virus infection.
We chose to replace SPI-2/CrmA with the baculovirus P35 gene since it has been shown
to inhibit apoptotic induction via caspase inhibition in a number of systems (250). In
each construct P35 was fused to the native SPI-2/CmrA promoter replacing the natural
SPI-2 or CrmA gene. The general strategy for recombinant virus construction is shown
in Figures 8 and 9. Briefly, plasmids containing P35 cloned between SPI-2/CrmA
flanking regions were transfected into RPVASPI-2::lacZ and CPVAcrmA::lacZ infected
cells. The resulting recombinant RPV and CPV viruses (RPVASPI-2::P35 and
CPVACrmA::P35) were selected by transient dominant selection (97) and screened for
loss of lacZ function.
Both recombinant viruses were analyzed by sequencing to confirm the presence
of P35 gene. Expression of recombinant protein by mutant viruses was analyzed by
immunoblot and is shown in Figure 16. P35 expressed in RPV migrate at 35 kDa and
began to appear at 2 hours post infection as indicated by the immunoreactive band
present only in RPVASPI-2::P35 but not in RPV or mock infected cell extracts (Fig.
16A). But surprisingly the levels of P35 in recombinant RPV infected cell extracts start
to decrease at 10 hours post infection and disappear altogether from immunoblots by 14
hours post infection (Fig. 16A, lanes 4-7).

131
P35, when expressed in the context of CPV, was seen to migrate at 35 kDa and
appear as a diffuse immunoreactive band present only in CPVAcrmA::P35 infections as
early as 2 hours post infection (Fig. 16B). But unlike in the case of RPVASPI-2::P35
infections (Fig. 16A), P35 expressed in CPVAcrmA::P35 was stable throughout the
course of infection up to 16 hours post infection (compare Fig. 16A and 16B, lanesl-
7). In order to ascertain if the differences in P35 expression seen within the context of
RPV and CPV infections are related to apoptosis, we analyzed infected cells for
morphological and biochemical features of apoptotic induction.
RPVASPI-2::P35 Induces Apoptosis in LLC-PK1 Cells
The reason for replacing SPI-2 in RPV with P35 was because P35 is a pan-
caspase inhibitor and functions to prevent the induction of apoptosis in a number of
diverse systems (250). It was rather unexpected to note that the levels of P35 decreased
after 8 hours of recombinant RPV infection (Fig. 16A, lanes4-7). To investigate if this
observation was related to apoptosis, we tested infected cells for morphological changes
associated with apoptosis (Fig. 12, E) and infected cell extracts for caspase activity
(Fig. 14).
DAPI staining of RPVASPI-2::P35 infected cells at 16 hours post infection
revealed the presence of densely staining apoptotic nuclei similar to those seen in RPV
and RPVASPI-2 infections (Fig. 12). This result implied that in the context of RPV, P35
was unable to block the induction of apoptosis. To test if the morphological observations
of infected cells correlated with biochemical activity of caspases within infected cells, we
analyzed infected cell extracts for the ability to cleave Ac-DEVD-AMC substrate (Fig.

132
14). Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Cell extracts are
incubated with Ac-DEVD-AMC and the rate of cleavage is determined fluorometrically.
RPV containing functional SPI-2 gene also induces caspase activity, although the rate of
peptide cleavage stabilizes after 9 hours post infection. The rate of cleavage of Ac-
DEVD-AMC in RPVASPI-2::P35 infections was seen to increase after 6 hours post
infection and peaked at 12 hours post infection although at levels slightly lower than that
seen in RPVASPI-2::lacZ infected extracts (Fig. 14). This result implies that P35 is
unable to function as an inhibitor of caspase activity within the context of RPV.
Comparing levels of P35 as seen in immunoblots, infected cells stained with DAPI
stained and caspase activity (Figs. 12, 14 and 16A), there seems to be a strong correlation
between the induction of apoptosis and stability of P35 during virus infection. These
results suggest that the reason RPVASPI-2::P35 infections of swine cells induce
apoptosis maybe due to the fact that P35 is unavailable to block apoptotic induction that
occurs typically after 6 to 8 hours of virus infection (Fig. 14). We devised experiments to
determine whether this event is specific to P35 or is a generalized phenomenon occurring
during RPV infections.
CPVAcrmA::P35 Blocks Apoptotic Induction in LLC-PK1 Cells
In order to investigate whether recombinant CPVAcrmA::P35 infected cells
display signs of apoptosis, infected cells were stained with DAPI and viewed
microscopically (Fig. 13). CPVAcrmA::P35 infected swine cells stained with DAPI had
similar morphological features as CPV infected cells and did not display characteristics
associated with apoptosis as seen with CPVAcrmA::lacZ infections (Fig. 13). This

133
A.
RPVASPI-2::P35
Hrsp.i 2 6 8 10 12 14 16 R M
B.
CPVAcrmA::P35
Hrs p.i
p35
Figure 16. Comparison of P35 expressed in RPV and CPV. LLC-PK1 cells infected
with either (A) RPVASPI-2::P35 or (B) CPVAcrmA::P35 were harvested at various times
post infection. Cell extracts were subjected to SDS-PAGE and immunoblotted with
polyclonal antisera to P35. Arrow indicates the position of migrating P35 proteins at 35
kDa. Abbreviations: R, wt RPV; M, Mock; C, wt CPV
8 10 12 14 16 C M

134
indicated that P35 was able to function like CrmA during CPV infections of pig kidney
cells. To confirm our findings with DAPI stained cells, we next assayed infected cell
extracts for the ability to cleave Ac-DEVD-AMC.
Cleavage of Ac-DEVD-AMC indicative of caspase activity is typically present in
cells undergoing apoptosis. Cell extracts are incubated with Ac-DEVD-AMC and the
rate of peptide cleavage was determined over time. Extracts from swine cells infected
with CPVAcrmA::P35 were able to block Ac-DEVD-AMC cleaving activity as in the
case of wt CPV infected extracts and unlike CPVAcrmA::lacZ did not induce caspase
activity (Fig. 15). This result confirmed that unlike in the case of RPVASPI-2::P35, P35
within the context of CPV was fully functional as a caspase inhibitor even after 8 hours
post infection (compare Fig. 14 and 15). Furthermore, P35 was able to substitute for
CrmA in blocking the induction of apoptosis during CPV infections. We did however
notice that the rate of caspase activity due to CPVAcrmA::P35 increased slightly after 14
hours post infection. However this slight increase did not result in any morphological
changes in infected cells since the DAPI stains (Fig. 13) were performed at 16 hours post
infection. It appears that P35 is inherently unstable when expressed within RPV but not
within CPV. If this were true, these results would further demonstrate the differences
between RPV and CPV, as P35 was able to function as a caspase inhibitor only in the
later case.
Viral Protein Expression: Effect of AraC
To determine if late events during viral replication have any influence on the
outcome of P35 expressed during RPVASPI-2::P35 infections as seen in Fig. 16A, we

135
analyzed P35 expression in the presence of cytosine arabinoside (AraC) (Fig. 17A).
AraC inhibits the synthesis of poxvirus DNA and thus prevents the expression of late
poxvirus proteins. Infected cell extracts prepared at early (6 hours) and late times (15
hours) post infection were immunoblotted for P35. As seen in Figure 17A, lanes 1 and 2,
P35 expressed in RPVASPI-2::P35 is present at 6 hours post infection both in the
presence and absence of AraC as expected although its expression in the presence of
AraC is increased. The increase in P35 can be explained by the ability of AraC to
prevent poxvirus DNA replication and thus late gene transcription leading to extended
early gene synthesis. At 15 hours post infection P35 was detected only in the presence of
AraC (Fig. 17A, lanes 3 and 4). However critical analysis of the data shows that the
relative decrease of P35 in the presence of AraC (Fig. 17A, lanes 1 and 4) is similar to
that in the absence of AraC (Fig. 17A, lanes 2 and 3), the only difference being higher
starting levels of P35 in the presence of AraC. This suggests that P35 is inherently
unstable within RPV infections and late events in viral replication may or may not be
responsible for decrease in P35 expression levels in RPVASPI-2::P35. Ac-DEVD-AMC
cleavage assays performed on extracts prepared from cells treated as mentioned reveal
that AraC blocks the induction of apoptosis (data not shown) indicating that apoptosis
occurs after/as a result of viral DNA replication.
At 6 hours post infection, P35 expressed in CPVAcrmA::P35 infections is seen
both in the presence and absence of AraC (Fig. 17B, lanes 1 and 2). However at 15 hours
post infection P35 expressed in the context of CPV is present even in the absence of
AraC (Fig. 17B, lanes 3 and 4) unlike in the case of RPVASPI-2::P35 infections (Fig.
17A, lanes 3 and 4). This clearly indicates that P35 is stable despite late events of virus

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A.
RPVASPI-2::P35
6 hr p.i 15 hr p.i
AraC
p35
+ + RPV
- 36 kDa
1 2 3 4 5
B.
CPVAcrmA::P35
6 hr p.i 15 hr p.i
AraC + + cpv
p35
- 36 kDa
1 2 3 4 5
Figure 17. Effect of AraC on P35 expression in RPV and CPV. LLC-PK1 cells were
infected with either (A) RPVASPI-2::P35 or (B) CPVAcrmA::P35 in the presence or
absence of AraC as indicated and harvested at early (6hr) or late times (15hr) post
infection. Cell extracts were subjected to SDS-PAGE and immunoblotted with
polyclonal antisera to P35. Arrow indicates the position of migrating P35 proteins at 35
kDa.

137
infection as seen in CPVAcrmA::P35. We conclude that the issue of P35 stability is
probably due to different biochemical events that occur during RPV but not CPV
infections.
P35 expression from within CPVAcrmA::P35 is stable and the recombinant
protein is capable of substituting for CrmA function in preventing caspase induction.
Therefore we chose to concentrate our efforts in using this recombinant virus to further
elucidate the in vivo role of CrmA during CPV infections of CAMs.
Recombinant CPV Viruses Expressing SERP2 or CrmA D303A
CPV infections of 11 day old CAMs produce red, hemorrhagic, non-inflammatory
pocks whereas deletion of the CrmA gene from CPV induces white, inflammatory pocks
with lower virus yields (306). Therefore CrmA functions to increase virus yields and
inhibit the induction of an inflammatory response on CAMs. Using the CAM model of
CPV infection, our experiments were directed to address two general questions. The first
was whether CrmA inhibits inflammation on CAMs by functioning as a protease
inhibitor. The second was whether the ability of CrmA to inhibit caspases (in particular
caspase-1) was sufficient to explain the ability of CrmA to control inflammation and
virus yield during CAM infections. We first produced two additional recombinant CPVs
in addition to CPVAcrmA::P35 in which either the critical PlAsp, key to caspase
recognition, was mutated (CPVCrmA D303A) or the entire CrmA gene was replaced by
the SERP2 of myxoma virus. Both P35 and SERP2 have been described as potent
inhibitors of caspases (314, 474). The general strategy for construction of these viruses is

138
illustrated in Figure 9. It is important to note that in each construct, the native CrmA
promoter regulated the relevant gene, CrmA D303A, P35 or SERP2.
The expression of the various proteins from the CrmA promoter of the
recombinant CPVs was analyzed by immunoblots of infected cell lysates (Fig. 18). The
data show expression of SERP2 (Fig. 184A) or CrmA D303A (Fig. 18B). SERP2 within
the context of CPV migrates at 34 kDa as expected (Fig. 184A, lane 1). In Fig. 184B,
lane 5, we note that CrmA from wild type CPV infections migrates as a 38 kDa protein,
whereas the CrmA D303A protein has a somewhat slower mobility resulting in a band
migrating at approximately 40 kDa (Fig.l8B, lane 7). We have noted altered
electrophoretic mobilities of serpins through mutations within the PI RCL mutations
previously for SPI-1 (Kristin Moon and Richard Moyer unpublished data) and SERP2
(Peter Turner and Richard Moyer unpublished data). The absence of CrmA
immunoreactive band in CPVAcrmA::lacZ (Fig. 18B, lane 6) authenticates this virus
construct. Each of the proteins analyzed appeared to be quite stable within the context of
CPV infected cells (data not shown).
Infections of LLC-PK1 Cells with CPV Recombinants: DAPI Stained Cells
It has been demonstrated that CrmA functions to inhibit caspase activation during
CPV infections of LLC-PK1 cells and thus prevents apoptotic induction in these cells.
Since the reason for replacing CrmA in CPV with other caspase inhibitors was to
establish CrmAs role during CPV infection, we first wanted to determine the outcome of
pig kidney cell infections in terms of caspase induction by the various recombinant
CPVs. We have already observed that P35 replacements of CrmA in CPV were able to

139
A.
1 2 3
Serp2*
34 kDa
Figure 18. Expression of SERP2 and CrmA D303A recombinant proteins. Extracts
were made from LLC-PK1 cells (A) or CV-1 cells (B) infected at an MOI of 10,
harvested at 16 hours post infection, subjected to SDS-PAGE and immunoblotted with
antisera to (A) Serp2 and (B) CrmA. Lanes: 1, CPVAcrmA::SERP2; 2, wt CPV; 3,
Mock; 4, Mock; 5; wt CPV; 6, CPVAcrmA::lacZ; 7, CPVCrmA D303A. Arrows indicate
the position of migrating proteins as follows: Serp2 34 kDa; CrmA -38 kDa and CrmA
D303A 40 kDa. Immunoblots indicate that the recombinant proteins are being
expressed from during virus infection.

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prevent the induction of apoptosis judged both by DAPI staining of infected cells and by
testing cell extracts for caspase activity (Fig. 13 and 15). Similarly, pig cells infected
with CPVAcrmA::SERP2 or CPVCrmA D303A were stained with DAPI, the results of
which are shown in Figure 19. The presence of densely staining apoptotic nuclei
indicated that CPVACrmA::SERP2 infections looked similar to CPVAcrmA::lacZ
infected cells (Fig. 19). This result indicates that SERP2 failed to inhibit the induction of
apoptosis during CPVAcrmA::SERP2 infections and that SERP2 was unable to substitute
for CrmA function in CPV, confirming previous reports (424). Thus SERP2 despite
being a caspase inhibitor, fails to block apoptotic induction during virus infection of
swine cells.
CPVCrmA D303A virus infections of pig kidney cells likewise were
indistinguishable from those of CPVAcrmA::lacZ (Fig. 19). The D303A RCL mutant
virus infected cells showed morphological signs of undergoing apoptosis such as the
presence of apoptotic bodies. This result indicates that CrmA functions as a protease
inhibitor during CPV infections to prevent the induction of apoptosis. The results
obtained from the D303A mutant virus underscores the importance of the PI position for
CrmA in the recognition of protease targets to prevent apoptotic induction. Since both
CPVAcrmA::SERP2 and CPVCrmA D303A viruses induce apoptosis in swine cells, we
expected to see caspase activity in extracts made from these infected cells. However P35
replacements of CrmA in CPV infections of LLC-PK1 cells were similar to wt CPV
infections and did not induce apoptosis as analyzed morphologically (Fig. 13) and
biochemically (Fig. 15).

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Infections of LLC-PK1 Cells with CPV Recombinants: Ac-DEVD-AMC Cleavage
Activity
To confirm biochemically, the morphological indications of apoptosis (Fig. 19),
we analyzed infected pig kidney cell extracts for the ability to cleave Ac-DEVD-AMC
(Fig. 20). Cleavage of Ac-DEVD-AMC is indicative of terminal caspase activity present
in cells undergoing apoptosis. Extracts were prepared at 12 hours post infection and
incubated in the presence of Ac-DEVD-AMC. Rates of Ac-DEVD-AMC cleavage were
calculated and are shown in Figure 20. As expected CPV infected extracts had little or
no peptide cleavage activity. CPVAcrmA::P35 extracts behaved like wt CPV extracts
and failed to cleave Ac-DEVD-AMC similar to results obtained in previous experiments
(Fig. 15).
However, CPVAcrmA::SERP2 and CPVCrmA D303A extracts behaved similarly
to CPVAcrmA::lacZ extracts and showed significant cleavage of the fluorogenic peptide
(Figure 20). These results correlate with DAPI stain results obtained in Figure 19 and
confirm the presence of caspase activity in CPVAcrmA::lacZ, CPVAcrmA::SERP2 and
CPVCrmA D303A infected cell extracts. Thus SERP2 is unable to replace CrmA to
prevent the induction of caspases during CPV infections of swine cells. Perhaps this is
due to species specificity of SERP2, i.e. a poor affinity of SERP2 for pig caspases (but
not rabbit caspases). Indeed SERP2 has been reported to function as an apoptotic
inhibitor during myxoma virus infections of the European rabbit (257). SERP2 has also
been shown to prevent apoptotic induction during myxoma infections of rabbit kidney
cells (Peter Turner and Richard Moyer, unpublished). Our results with CrmA D303A
mutant virus further confirm the importance of the RCL in CrmA function and also

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A. Mock
B. CPV
C. CPVAcrmA::SERP2 D. CPVCrmA D303A
Figure 19. Morphological changes in LLC-PK1 cells infected with
CPVAcrmA::SERP2 or CPVCrmA D303A. Pig kidney cells were infected at MOI 10
and stained with DAPI at 16 hours post infection. Panels indicate the different viruses
used for infection (A) Mock, (B) CPV, (C) CPVAcrmA::SERP2 and (D) CPVCrmA
D303A. Examples of apoptotic bodies, characteristic of cells undergoing apoptosis are
indicated by arrowheads.

1 2 3 4 5 6
Figure 20. Biochemical changes in LLC-PK1 cells infected with
CPVAcrmA::SERP2 or CPVCrmA D303A. Pig kidney cells were infected at MOI 10
and harvested at 12 hours post infection. Cell extracts were incubated with terminal
caspase substrate Ac-DEVD-AMC, a fluorogenic peptide. Cleavage of Ac-DEVD-
AMC is indicative of caspase activity. Ac-DEVD-AMC cleavage rates were determined
and are expressed arbitrarily as fluorescence signal units per second. Bar numbers: 1,
Mock; 2, CPV; 3, CPVAcrmA::lacZ; 4, CPVAcrmA::P35; 5, CPVAcrmA::SERP2; 6,
CPVCrmA D303A.

144
strengthens the hypothesis that CrmA acts as a protease inhibitor to prevent the activation
of caspases.
Recombinant CPV Infections of CAMs
During CPV infections of CAMs, CrmA is presumed to act directly on caspase-1
to prevent the maturation of inflammatory cytokines IL-1P and IL-18 from their
respective precursors. If this hypothesis were true, then replacing CrmA in CPV with
other caspase inhibitors should produce similar results as wt CPV. We chose pan-
caspase inhibitor P35 based on the ability of P35 to inhibit caspase activation in a number
of species (250). Indeed P35 is able to function as caspase inhibitor and as a CrmA
replacement during CPV infections of LLC-PK1 cells, therefore we expected P35 to
functionally replace CrmA in CPV infections of CAMs.
Second, we chose SERP2 as a replacement for CrmA in CPV to answer the
question of whether poxvirus serpins from different poxviruses had similar functional
targets. If this were true, we would expect SERP2 to functionally replace CrmA in CPV.
Considering that SERP2 failed to inhibit caspase induction within the context of CPV
infections of swine cells, we were unsure of what to expect during CAM infections using
the various CPV derivatives.
Third, the ability of CrmA to inhibit an inflammatory response during CPV
infections of CAMs may not be related to CrmAs ability to act as a protease inhibitor,
more specifically a caspase inhibitor. The CrmA D303A mutation in the RCL alters
target protease specificity. Indeed CPVCrmA D303A is unable to block the activation of
caspases in pig cells and therefore is an excellent candidate to answer the last hypothesis.

145
Pock Morphology of Recombinant CPV Infected CAMs
CPV produces red, hemorrhagic pocks (Fig. 2IB), while CPVAcrmA::lacZ virus,
from which CrmA was deleted, produces white pocks as expected (Fig. 21C) (306). The
white pocks produced by CPVAcrmA::lacZ also turn dark blue in the presence of NBT
indicating the presence of activated heterophil influx, consistent with previous reports
(306). Interestingly, the CrmA D303A mutant virus, in which the PI Asp was mutated,
produced white inflammatory pocks (Fig. 2 IF). This result suggests that the PI Asp
within the reactive center loop is required for CrmA to inhibit inflammation in vivo and
further that CrmA inhibits inflammation by functioning as a protease inhibitor.
Surprisingly, both P35 and SERP2 recombinant viruses produced white pocks containing
activated heterophils as suggested by positive NBT staining (Fig. 21D and 2IE).
Therefore, P35 and SERP2 fail to inhibit the inflammatory response during CPV
infections of CAMs. This was an unexpected result since both P35 and SERP2, like
CrmA have been shown to inhibit caspase-1 in vitro, which should serve to control the
processing of IL-P and IL-18, both of which are inflammatory cytokines.
All White Pocks Contain Heterophils
Histological analysis of pocks was performed in order to confirm that white pocks
which stained positive with NBT (Fig. 21C to F) were indeed inflammatory. Individual
pocks were isolated, sectioned and stained with hematoxylin and eosin. The sections
viewed microscopically are shown in Figure 22. CPV infected pocks exhibit hemorrhage
between the ectoderm and mesoderm with few heterophils (Fig. 22C and 22D).

146
-NBT
+NBT
A.
C.
Figure 21. Pock morphology on CAMs infected with recombinant viruses. Pock
formation by (A) Mock, (B) CPV, (C), CPVAcrmA::lacZ, (D) CPVAcrmA::P35 (E)
CPVAcrmA: :SERP2 and (F) CPVCrmA D303 A on the CAMs of 11 day old chicken
embryos, 3 days post infection. Membranes or sections of membranes were untreated or
stained with NBT to show the presence of activated heterophils indicated by dark staining
of pocks.

147
-NBT
+NBT
Figure 21. Continued

148
Hyperplasia of the ectoderm coincides with regions of pock formation. The pocks from
CPVAcrmA::lacZ infections also showed extensive inflammatory cell influx into the
mesoderm, most of which were heterophils, similar to previous reports (Fig. 22E and
22F) (306). The pock histology from CPVCrmA D303A infections appeared nearly
identical to that of CPVAcrmA::lacZ (Fig. 22K and 22L). The pocks of CPVAcrmA::P35
and CPVAcrmA::SERP2 were also very similar to those of CPVAcrmA::lacZ but had
even greater levels of heterophils (Fig. 22G to J). Therefore it is clear that P35 and
SERP2 cannot block infection-induced inflammation on the CAM.
Inflammatory Pocks Show High Levels of NADPH Oxidase
Activated chicken heterophils have been shown to contain NADPH oxidase
activity that produces superoxide radicals which can be measured by the reduction of
tetrazolium salts (256). We compared the levels of NADPH oxidase within pocks by
measuring the reduction of 3,(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium (MTT),
within infected membranes as shown in Figure 23. CPV infected membranes showed
activity only slightly higher than mock infected membranes. Significantly higher activity
was observed from the white pocks produced by each of the recombinant viruses lacking
a functional CrmA gene but there were only slight differences in activity between the
various inflammatory pock infections (Fig. 23). These results confirm that the
heterophils seen in all the white pocks were indeed activated.

149
Figure 22. Histological examination of pocks from infected CAMs. CAMs infected
with recombinant viruses were harvested 3 days post infection. Individual pocks were
sectioned and stained with hematoxylin and eosin. (A) Mock infected CAMs consist of a
thin ectoderm, approximately 2 cells in thickness, a mesoderm with low numbers of cells
in the extracellular matrix and a thin endoderm, 1-2 cells thick. (B) Mock infected
CAMs featuring individual mesodermal cells at 500 x magnification. (C) CPV infected
pocks show marked ectodermal and mesodermal hyperplasia. Hemorrhage can be seen
between ectodermal and mesodermal layers of the CAM. (D) CPV infected pocks
showning the ectodermal and mesodermal junction at 500 x magnification. (E & F)
CPVAcrmA: :lacZ, (G & H) CPVAcrmA::P35, (I & J) CPVAcrmA::SERP2 and (K & L)
CPVCrmA D303A pocks show ectodermal hyperplasia and extensive heterophilic
inflammation in the mesoderm without evidence of hemorrhage. H-heterophils
(indicated by arrows); E-Ectoderm; M-mesoderm.

150
Figure 22. Continued

151
Figure 23. Inflammatory pocks show similar levels of oxidative burst. Confluently
infected CAMs were harvested at 48 hours post infection. Reactive oxygen intermediates
were measured by incubating pooled pocks in the presence of MTT for 30 minutes. The
reduced formazan was dissolved in DMSO and absorbance read at 550nm. The results
are expressed as OD 550/mg of tissue. Endogenous NADPH oxidase activity from
uninfected CAMs have been subtracted. Bar numbers: 1, Mock; 2, CPV; 3,
CPVAcrmA::lacZ; 4, CPVAcrmA::P35; 5, CPVAcrmA::SERP2; 6, CPVCrmA D303A.
The results are standard error of the mean of four such experiments. denotes P < 0.05
by Students t test compared to CPV.

152
P35 and SERP2 Restore Viral Yields from Infected CAMs
When compared to pocks produced by wt CPV, the pocks produced by
CPVAcrmA::lacZ are inflammatory and have been shown to produce far less virus (306).
It was presumed that the reduction in virus yields was due to the induction of an
inflammatory response in the CAMs. If this were true we would predict that all the
inflammatory pocks produced by the various recombinant CPV should also have lowered
virus yields. We have measured the ability of P35 and SERP2 to restore virus yields.
Following infection, individual pocks were harvested, the pocks homogenized and the
virus titers determined on CV-1 cells. The resulting virus yields were expressed as plaque
forming units (PFU)/pock (Fig. 24). Wild type CPV pocks yield approximately 5 x 106
PFU/pock, while CPVAcrmA::lacZ yields were at least a ten fold lower (4 x 105
PFU/pock) consistent with previous reports (306). Yields of virus from CPVCrmA
D303A pocks were similar to those of CPVAcrmA::lacZ, a result again consistent with
the prediction that the PI Asp of the RCL is required for CrmA to function as a protease
inhibitor during CPV infection in vivo. Both P35 and SERP2 recombinant viruses are
able to restore virus yields to wild type levels. Hence the two proteins P35 and SERP2
can partially substitute for CrmA function within the context of a CPV infection. These
results show that the reduction in pock titers and the white, inflammatory pock phenotype
are not linked and reflect two different functions of CrmA. The restoration of virus
yields, despite the presence of inflammation, suggests that CrmA, might function within
the CAM to control apoptosis and that induction of apoptosis, rather than inflammation in
the absence of CrmA leads to lowered virus yields. It is important to note that in cell

153
culture there is little effect of CrmA on virus yield as virus produced by wt CPV and
CPVAcrmA are indistinguishable (332).
P35, SERP2 and CrmA Block Induction of Terminal Caspase Activity within
Infected Pocks
Inhibition of apoptosis by CrmA in CPV infected cells in culture has been
previously demonstrated (332). Based on the known inhibitory profiles of CrmA, P35
and SERP2 towards apical caspases (caspases -8 and -10), which are involved in
apoptosis, SERP2 and P35 might also be expected to inhibit apoptosis of infected cells in
vivo as well as in cell culture. Additional evidence that P35 and SERP2 inhibit induction
of terminal caspase activity and block apoptosis can be seen when these viruses are used
to infect primary chicken embryo fibroblasts (CEFs). While no induction of terminal
caspase activity was noted with CPV, CPVAcrmA ::P3 5 or CPVAcrmA ::SERP2
infections, CPVAcrmA: :lacZ induced terminal caspase activity during infection of CEFs
(Fig. 25). However, we have observed that in cell culture, involving pig kidney cells,
only P35 functions like CrmA and is able to block the induction of apoptosis within the
context of CPV infections (Fig. 13 and 15). SERP2 within the context of CPV infections
fails to prevent the induction of apoptosis in swine cells (Fig. 19 and 20).
In order to determine if infected pocks contain activated caspases, we have
assayed infected pock extracts for the ability to cleave Ac-DEVD-AMC, a fluorogenic
peptide (Fig. 26). Cleavage of Ac-DEVD-AMC is indicative of terminal caspase activity
and induction of apoptosis. While little caspase induction within pocks is noted in CPV
infections (similar to mock extracts), both CPVAcrmA: :lacZ and CPVCrmA D303A

154
u
o
c.
p
Lu
Cu
107
Figure 24. P35 and SERP2 restore viral yields from infected CAMs. Individual
pocks from infected 11 day old CAMs were isolated at 3 days post infection. Pock
homogenates were plaque assayed on CV-1 cells. Viral plaques were counted 3 days
later. The results are expressed as plaque forming units (PFU)/pock and are averages of
viral titers from 12 pocks. Bar numbers: 1, CPV; 2, CPVAcrmA::lacZ; 3,
CPVAcrmA::P35; 4, CPVAcrmA::SERP2; 5, CPVCrmA D303A. Error bars are standard
deviations of the mean. denotes P < 0.05 by Students t test compared to CPV.

155
infections induce terminal caspase activity to similar levels (Fig. 26). This induction of
terminal caspase activity by infection with CPVCrmA D303A reaffirms that the PI Asp of
the RCL is an essential feature of CrmA function and is required to prevent caspase
induction. Both P35 and SERP2, like CrmA, in the context of CPV, block induction of
terminal caspase activity within pocks (Fig. 26).
The ability of CPVAcrmA::SERP2 to prevent the induction of avian caspases in
both pock and CEF extracts contrasts the results we obtained with similar infections of
swine cells (Fig. 20). These results suggest that the restoration of pock titers seen in both
P35 and SERP2 recombinant infections is due to preventing apoptosis. These results also
imply that the reduction in virus yields seen in CPVAcrmA::lacZ and CPVCrmA D303A
infections were due to a failure to prevent apoptosis and not due to containment by
inflammation and/or the presence of activated heterophils. Thus CrmA appears to control
both inflammation and apoptosis within CAMs and P35 and SERP2 can substitute for
CrmA only to control apoptosis but not inflammation. Since all three proteins act to
inhibit caspases, the possibility exists that CrmA inhibits inflammation by a novel non-
caspase dependent mechanism.
Effect of Caspase Inhibitors on Protease Activity Present in Infected CAMs
Based on the ability to cleave Ac-DEVD-AMC, CAM extracts from
CPVAcrmA::lacZ and CPVCrmA D303A contain a putative caspase-3-like activity (Fig.
26). We then determined the range of caspase activity within CAMs with the use of
caspase specific peptide inhibitors. The peptide Ac-WEHD-CEIO is an inhibitor specific
for human caspase-1, whereas Ac-DEVD-CHO is a potent peptide inhibitor for human

156
hr p.i.
Figure 25. P35, SERP2 and CrmA block terminal caspase activity within CEF cells.
CEF cells were infected at MOI10 and harvested at various times post infection. Cell
extracts were made and incubated with terminal caspase substrate Ac-DEVD-AMC, a
fluorogenic peptide. Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Ac-
DEVD-AMC cleavage rates were determined at each harvested time point and are
expressed arbitrarily as fluorescence signal units (FSU) per second. Legends
corresponding to the different viruses tested are indicated.

157
Figure 26. P35, SERP2 and CrmA block terminal caspase activity within infected
pocks. Individual pocks from infected 11 day old CAMs were isolated at 3 days post
infection. Lysates of individual pocks were made and protein concentrations determined.
25 pg of extract was tested for the ability to cleave the substrate Ac-DEVD-AMC. The
results are expressed as rates of fluorescence per second and are averages of assays from
20 individual pocks (10 individual pocks tested for CPVCrmA D303A). Bar numbers: 1,
Mock; 2, CPV; 3, CPVAcrmA::lacZ; 4, CPVAcrmA::P35; 5, CPVAcrmA::SERP2; 6,
CPVCrmA D303A. The error bars are standard deviations of the mean. denotes PO.05
by Students t test compared to CPV. Insert: Eleven day old CAMs were infected
confluently with CPVAcrmA::lacZ. Extracts were made from infected CAMs harvested
at 48 hr post infection. 70 pg of protein from CPVAcrmA::lacZ CAM extracts was
preincubated with increasing concentrations of either Ac-WEHD-CHO (- -) or Ac-
DEVD-CHO (- -) at 37C for 2 hours. Residual caspase activity was then determined
by hydrolysis of Ac-DEVD-AMC, a fluorogenic substrate for caspase-3. Ac-DEVD-
AMC cleavage rates are expressed arbitrarily as fluorescence signal units (FSU) per
second.

158
caspase-3, -7 and -8 and Z-VAD-FMK is an inhibitor of all caspases (118). The
minimum concentrations of Ac-WEHD-CHO and Ac-DEVD-CHO determined to give
complete inhibition of human recombinant caspase-1 (1U) and caspase-3 (10U) was
determined to be lOOnM and lOnM respectively based on hydrolysis of fluorogenic
substrates (data not shown).
Extracts were made from confluently infected CAMS and pre-incubated for 2 hrs
in the presence of increasing concentrations of either Ac-WEHD-CHO or Ac-DEVD-
CHO and thereafter assayed for caspase 3-like activity as in Figure 26 using the
fluorogenic substrate Ac-DEVD-AMC (Fig. 26 Insert). The caspase-1 inhibitor, Ac-
WEHD-CHO up to lOOnM has no effect on the cleaving activity present in the extracts
(data not shown), which was not surprising since the substrate used in these reactions,
Ac-DEVD-AMC does not measure caspase-1-like activity. However, the caspase-3-like
inhibitory peptide, Ac-DEVD-CHO gave marked inhibition of the activity as expected.
No cleavage activity in these extracts was observed when the caspase-1-like substrate,
Ac-WEHD-AMC is used in comparable assays, indicating an absence of detectable
levels of caspase-1-like activity (data not shown). Therefore, based on these assays, all
caspase activity observed in extracts of CPVAcrmA::lacZ infected CAMs, are solely due
to caspase-3-like activity.
P35 and SERP2 are able to function like CrmA to inhibit the induction of
apoptosis and restore virus yields within CAMs; however, unlike CrmA, fail to inhibit
inflammation. Therefore the replacement of CrmA by P35 and SERP2 can only partially
complement CrmA function. Thus, the regulation of inflammation and apoptosis by
CrmA are two distinct properties, and may not depend on caspase inhibition alone.

159
Chicken ProIL-ip Processing Activity in CAMs.
Caspase-1 is responsible for the maturation of IL-ip and IL-18 (both are
inflammatory cytokines) from their respective precursors. The only infected CAM
extracts in which we observed any caspase activity, was in either CPVAcrmA::lacZ or
CPVCrmA D303A. Within those extracts, all the caspase activity we observed within
infected CAMs (Fig. 26 Insert) resembled caspase-3, consistent with the inability of these
viruses to control apoptosis. We have not been able to detect caspase-1-like activity
within infected CAMs using peptide substrates. However, a more sensitive assay for
caspase-1-like activity is cleavage of radiolabeled proIL-ip or proIL-18 precursor
molecules to active cytokines. Like CrmA, SERP2 and P35 also inhibit caspase-1.
Caspase-1 is believed to be involved in the processing of proIL-ip or proIL-18 cytokine
precursors through cleavage of the pro-inflammatory cytokine precursors at conserved
aspartate residues (99). The fact that CPVAcrmA::SERP2 and CPVAcrmA::P35 viruses
fail to control inflammation casts some doubts that inflammation is caspase mediated.
Examination of the avian and mammalian gene sequences raised further questions
in this regard. The activation of mammalian proIL-ip is characterized by caspase-1
mediated cleavage at Asp27 followed by cleavage at Asp 116, sites generally conserved
in mammals including humans (395). However, inspection of the chicken proIL-ip gene
showed both these critical aspartic acids to be missing (446). Although there are
neighboring aspartic acids present, they are not within the conserved caspase-1 cleavage
site context.
A cDNA clone of chicken IL-1 (3 which encoded amino acids 106 to the C-
terminus, was found to be active (446). The molecule was designed based on the

160
assumption that the N-terminal portion of chicken proIL-1 P, like mammalian proIL-ip,
was removed by processing during the activation process. These observations would be
consistent either with an alternative caspase-independent mechanism of processing, or a
somewhat different cleavage recognition site for avian caspase-1. However, in the case
of proIL-18, the vertebrate cleavage site is completely conserved within the known
chicken proIL-18 sequence (354). Given the known proinflammatory functions of IL-1 (3
and IL-18, we sought to determine whether any proIL-ip or proIL-18 processing activity
was detected within infected CAMs.
Despite the absence of the two critical aspartic acid residues, we first tested the
ability of infected CAM extracts to process 35S labeled chicken proIL-ip (Fig. 27).
Lysates were prepared from infected membranes and incubated with 35S radiolabeled
chicken proIL-1 p. Processing activity was measured by monitoring the cleavage of the
precursor molecules with SDS-PAGE and subsequent autoradiography. Precursor
chicken IL-1P migrated at approximately 29 kDa. The recombinant human caspase-1
(1U), as expected, based on the sequences (446), failed to process the chicken precursor
(Fig. 27, lane 1). More importantly, we also failed to see any processing of chicken
proIL-1 p by any of the infected CAM extracts (Fig. 27, lanes 2-6). This result suggested
two possibilities. First, the possibility that chicken IL-1 P is not involved in the
inflammation on CAMs. Second possibility is that if chicken IL-1 p were to be involved
in the inflammation on CAMs, the chicken IL-1P precursor itself is biologically active.

161
1 2 3 4 5 6
Figure 27. Chicken proIL-P processing. S labeled chicken proIL-ip proteins
synthesized in vitro were incubated with either buffer (lane 1), 1U of human caspase-1
(lane 1) or with 200 pg of extracts from confluently infected CAMs harvested 48 hours
post infection. Lanes: 1, buffer or 1U caspase-1; 2, CPV; 3, CPVAcrmA::lacZ; 4,
CPVAcrmA::P35; 5, CPVAcrmA::SERP2; 6, CPVCrmA D303A. The protein mixtures
were resolved by electrophoresis on 10% SDS-PAGE and visualized by autoradiography.
Radiolabeled peptides are: chicken proIL-ip 29 kDa.

162
Processing of ProIL-ip can be Blocked by either SERP2 or P35 within
Inflammatory Pocks
To ensure that our assay to detect caspase-1 activity was valid, we used an
alternative substrate such as mouse proIL-P in order to assay the chicken enzyme activity.
Extracts from infected CAMs were incubated with 35S radiolabeled mouse proIL-ip in
the presence or absence of recombinant human caspase-1. Processing activity was
measured by monitoring the cleavage of the precursor molecules with SDS-PAGE and
subsequent autoradiography. As seen in Figure 28, lane 1, native mouse proIL-lp
migrates as a peptide of 31 kDa. The mouse IL-1 p is processed as anticipated to an
intermediate 29 kDa peptide and then to the mature 17 kDa peptide by addition of human
caspase-1 (Fig. 28, lane 1). Again however, none of the infected CAM extracts were able
to process mouse proIL-ip (Fig. 28, lanes 2-6) to the mature form. We did, however, see
some partial processing of the mouse substrate with extracts from every infection except
wt CPV. Extended incubation (up to 6 hours) of the mouse proIL-1 P substrate with
CAM extracts did not yield mature IL-1 P from any infection condition (data not shown).
It is possible that the ability of CAM extracts to very minimally mediate partial
processing of mouse proIL-1 P but fail to completely maturate the mammalian cytokine
may be due to species specificity of the avian enzyme, i.e. a poor affinity of chicken
caspases for mouse proIL-ip. However, the question of species specificity could be
addressed using chicken proIL-18 as a substrate for chicken caspase-1 (see below).
Therefore within the limits of detectability, we found no evidence for the presence of any
caspase-1 activity in the infected CAMs against either mammalian or chicken proIL-ip.

Caspase-1
163
Figure 28. P35 and SERP2 function like CrmA to prevent Caspase-1 mediated
processing of proIL-ip. 35S labeled mouse proIL-ip proteins synthesized in vitro were
incubated with either buffer (lane 1), 1 U of human caspase-1 (lane 1) or with 200 jig of
extracts from confluently infected CAMs harvested 48 hours post infection. Similar
amounts of the protein extracts were also pre-incubated with 1U of human caspase-1
prior to the addition of mouse proIL-1 p. The protein mixtures were resolved by
electrophoresis on 10% SDS-PAGE and visualized by autoradiography. Lanes: 1, Buffer
or 1U caspase-1; 2, CPV; 3, CPVAcrmA::lacZ; 4, CPVAcrmA::P35; 5,
CPVAcrmASERP2; 6, CPVCrmA D303A. Radiolabeled peptides are: mouse proIL-ip -
31 kDa; intermediate 29 kDa; mature IL-1 p 17 kDa.

164
When exogenous human caspase-1 was added to extracts from CPVAcrmA::lacZ and
CPVCrmA D303A infected CAMs, cleavage and maturation of mouse IL-ip was noted
(Fig. 28, lanes 3 and 6). However, when caspase-1 was added to extracts from CPV,
CPVAcrmA::P35 and CPVAcrmA::SERP2 infections (Fig. 28, lanes 2, 4 and 5), no
processing of mouse proIL-1p to mature IL-ip was noted implying that there remains
sufficient amounts of functional P35 and SERP2 respectively in those extracts to inhibit
the added caspase-1. Therefore, P35 and SERP2 would function like CrmA to prevent
caspase-1 activity were caspase-1 present adding further evidence to the theory that
caspase-1 mediated IL-1 p activation may not be the cause of inflammation on CAMs
during CPV infection.
Processing of Chicken ProIL-18 can be Blocked by either SERP2 or P35 within
Inflammatory Pocks
Unlike proIL-ip, chicken proIL-18 maintains the mammalian proIL-18 cleavage
site. Therefore we also tested the ability of CAM extracts to cleave radiolabeled chicken
proIL-18 (Fig. 29). Lysates were prepared from infected membranes and incubated with
35S radiolabeled chicken proIL-18. Processing activity was measured by the cleavage of
the precursor molecules monitored following separation of the products by SDS-PAGE
and subsequent autoradiography. Radiolabeled, control chicken proIL-18 migrated at
approximately 23 kDa (Fig. 29, lane 1). When incubated with 1U of recombinant human
caspase-1, the precursor proIL-18 peptide was processed to the mature cytokine, which
migrated at 19 kDa on SDS-PAGE (Fig. 29, lane 2) indicating that the cleavage site is

165
Chicken
Pro-IL-18
Processed
Figure 29. P35 and SERP2 function like CrmA to prevent processing of chicken
proIL-18 in CAMs. 3iS labeled chicken proIL-18 proteins made in vitro were incubated
with either buffer (lane 1), 1U of human caspase-1 (lane 2) or with 200 pg of extracts
from confluently infected CAMs harvested 48 hours post infection. Lanes: 1, Buffer; 2,
1U caspase-1; 3, CPV; 4, CPVAcrmA::lacZ; 5, CPVAcrmA::P35; 6,
CPVAcrmA::SERP2; 7, CPVCrmA D303A. The protein mixtures were resolved by
electrophoresis on 10% SDS-PAGE and visualized by autoradiography. Radiolabeled
peptides are: chicken proIL-18 23 kDa; processed form 19 kDa.

166
recognized by human caspase-1. No cleavage was observed when proIL-18 was
incubated with extracts from CPV infected CAMs (Fig. 29, lane 3). However,
CPVAcrmA::lacZ and CPVCrmA D303A infected CAM extracts both contain an activity
able to cleave chicken proIL-18 (Fig. 29, lanes 4 and 7), whereas extracts from CPV,
CPVAcrmA::P35 or CPVAcrmA::SERP2 infections failed to process chicken proIL-18
(Fig. 29, lanes 3, 5 and 6). We conclude that P35 and SERP2 function like CrmA in
preventing chicken proIL-18 processing activity even though the respective recombinant
viruses still produce inflammatory pocks. Therefore IL-18, like IL-ip is unlikely to be
processed in this system. Hence, neither cytokine is likely to be the mediator of
inflammation on CAMs caused by either CPVAcrmA::P35 or CPVAcrmA::SERP2
infections.
Chicken ProIL-18 Processing Activity in CPVAcrmA Lysates is Blocked by Caspase
Specific Peptide Inhibitors.
Although collectively among the viruses tested there is no correlation with control
of inflammation and proIL-1 p or proIL-18 cleavage, extracts from CPVAcrmA:: lacZ and
CPVCrmA D303A virus infected CAMs do contain an activity which can process proIL-
18. We sought to provide further evidence that the processing activity is not related to
caspase-1. Therefore, we first assayed the ability of chicken proIL-18 to be cleaved by
purified caspases-1 or -3 and also verified the specificity of caspase inhibitors towards
caspase-1 (Fig. 30). As expected, chicken proIL-18 was readily cleaved by caspase-1.
Furthermore, that reaction was blocked by the caspase-1 inhibitor Ac-WEHD-CHO and
the general caspase inhibitor Z-VAD-FMK but unaffected by the caspase-3 inhibitor Ac-

167
DEVD-CHO (Fig. 30, lanes 1-5). Unlike previous reports (122, 133) we found little if
any indication for cleavage of proIL-18 by up to 15U of purified human caspase-3 to this
reaction (Fig. 30, lanes 6-9).
The inability of human caspase-3 to process chicken proIL-18 could be explained
due to species specificity, i.e. a low affinity of human caspase-3 for the chicken substrate.
However, we have also tested the ability of human caspase-3 to cleave mammalian
proIL-18 and found that up to 10U of recombinant human caspase-3 was unable to cleave
bovine proIL-18 (data not shown). Therefore the results in Figure 30 indicate that the
peptide inhibitors Ac-WEHD-CHO and Ac-DEVD-CHO were specific for caspases-1
and -3 respectively and that caspase-1 mediated cleavage of chicken proIL-18 is inhibited
by Ac-WEHD-CHO but not Ac-DEVD-CHO.
We then re-investigated the proIL-18 cleaving activity observed in the extracts
from CPVAcrmA::lacZ infected CAMs. Extracts were preincubated with the peptide
inhibitors prior to the addition of radiolabeled chicken proIL-18 (Fig. 31). Native
chicken proIL-18 migrates as a 23 kDa polypeptide (Fig. 31, lane 1). CPVAcrmA::lacZ
CAM extracts cleave chicken proIL-18 to the 19 kDa mature peptide (Fig. 31, lane 2).
Preincubation of extracts from CPVAcrmA::lacZ infections with Ac-DEVD-CHO or Z-
VAD-FMK but not Ac-WEHD-CHO blocked the processing of chicken proIL-18 (Fig.
31, compare lanes 2-5). Despite a failure of purified caspase-3 to cleave chicken proIL-
18 (Fig. 30), these results are most consistent with a terminal caspase mediated cleavage
of proIL-18. In addition to caspase-1 mediated processing of proIL-18, there also exist
alternative processing mechanisms to yield biologically active cytokine (122, 133, 389).
Therefore an alternative, a non-caspase enzyme inhibited by both Ac-DEVD-CHO and

168
Chicken
Pro-IL-18
Processed
1 2 3456789
Figure 30. Caspase-1 mediated processing of chicken proIL-18 is blocked by
peptides. 35S labeled chicken proIL-18 proteins synthesized in vitro were incubated with
either buffer (lane 1), 1U of human caspase-1 (lane 2), caspase-1 preincubated with 100
nM Ac-WEHD-CHO (lane 3), caspase-1 preincubated with 10 nM Ac-DEVD-CHO
(lane 4), caspase-1 preincubated with 200 pM Z-VAD-FMK (lane 5), 15U of human
caspase-3 (lane 6), caspase-3 preincubated with 100 nM Ac-WEHD-CHO (lane 7),
caspase-3 preincubated with lOnM Ac-DEVD-CHO (lane 8) or caspase-3 preincubated
with 200 pM Z-VAD-FMK (lane 9). The protein mixtures were resolved by
electrophoresis on 10% SDS-PAGE and visualized by autoradiography. Radiolabeled
peptides are: chicken proIL-18 23 kDa; processed form 19 kDa

169
1 2 3 4 5
6 7 8 9 10
Figure 31. Chicken proIL-18 processing activity in CPVAcrmA::lacZ is inhibited by
caspase specific peptides. 35S labeled chicken proIL-18 synthesized in vitro was
incubated with either buffer (lane 1), 200 pg of CPVAcrmA::lacZ extracts from
confluently infected CAMs harvested 48 hours post infection (lane 2), CPVAcrmA::lacZ
extracts preincubated with 100 nM Ac-WEHD-CHO (lane 3), CPVAcrmA::lacZ extracts
preincubated with 10 nM Ac-DEVD-CHO (lane 4), CPVAcrmA::lacZ extracts
preincubated with 200 pM Z-VAD-FMK (lane 5), CPVAcrmA::lacZ extracts alone (lane
6), CPVAcrmA::lacZ extracts preincubated with 1U of human caspase-1 (lane 7),
CPVAcrmA::lacZ extracts preincubated with 100 nM of Ac-WEHD-CHO and caspase-1
(lane 8), CPVAcrmA::lacZ extracts preincubated with 10 nM Ac-DEYD-CHO and
caspase-1 (lane 9) or CPVAcrmA::lacZ extracts preincubated with 200 pM Z-VAD-
FMK and caspase-1 (lane 10). The protein mixtures were resolved by electrophoresis on
10% SDS-PAGE and visualized by autoradiography. Radiolabeled peptides are: chicken
proIL-18 23 kDa; processed forms 19 kDa.

170
Z-VAD-FMK may be responsible for proIL-18 processing in CAMs infected with
CPVAcrmA::lacZ.
The results of Figs. 29 and 31 suggest that the chicken proIL-18 processing
activity present in CPVAcrmA::lacZ infections is not due to caspase-1 but rather a
terminal caspase-3-like enzyme present in the apoptotic extracts. In order to eliminate
the possibility that the extracts might contain an endogenous caspase-1 inhibitor, we
added exogenous caspase-1 to CPVAcrmA::lacZ lysates and assayed for chicken proIL-
18 processing activity either in the presence or absence of peptide substrates (Fig. 31,
lanes 7-10). Samples containing exogenously added caspase-1 activity together with the
endogenous caspase-3-like activity present in the CAM extract, fully processed precursor
IL-18 to the mature form (Fig. 31, lane 7). Comparing lanes 7 to 10 in Figure 31, we
concluded that exogenously added caspase-1 activity and any endogenous caspase
activity in the CPVAcrmA::lacZ extracts could be blocked by Z-VAD-FMK (Fig. 31,
lane 10). In the presence of Ac-WEHD-CFIO, which inhibits caspase-1, the remaining
caspase-3-like activity in the extracts remained and produced cleavage (Fig. 31, lane 8).
Conversely Ac-DEVD-CHO blocked caspase-3-like activity in the CPVAcrmA::lacZ
CAM extracts, though the remaining exogenously added caspase-1 activity results in
chicken proIL-18 cleavage (Fig. 31, lane 9). We conclude that the chicken proIL-18
processing activity seen in CPVAcrmA::lacZ extracts is due to a terminal caspase-3-like
activity and not due to caspase-1. Furthermore the white, inflammatory pocks generated
from CPVAcrmA::P35 and CPVAcrmA::SERP2 are not likely the result of caspase-1
mediated proIL-lp or proIL-18 cleavage.

171
Caspase-3 Activity in the Presence of CAM Extracts
In order to eliminate the possibility that the extracts might contain an endogenous
caspase-3 inhibitor, we added exogenous caspase-3 to CPVAcrmA::lacZ lysates and
assayed for the ability of the protein mixtures to cleave the caspase-3 substrate Ac-
DEVD-AMC fluorometrically. As seen in Figure 32 when 10U of recombinant human
caspase-3 was added exogenously to CPVAcrmA::lacZ extracts, there was an increased
effect on the rate of Ac-DEVD-AMC cleavage that was due to the combined activities of
either caspase-3 or extracts tested alone. From this result we find no evidence of
CPVAcrmA::lacZ infected CAM extracts to inhibit caspase-3 and therefore conclude that
the chicken proIL-18 processing ability found in the extracts seen in Figure 32 to be due
to a caspase-3-like enzyme.
However, it was interesting to note that both P35 and SERP2 replacements of
CPV induced an inflammatory response on the CAMs but yet extracts from these
infections failed to process proIL-1p or proIL-18. This observation suggest that IL-1 p or
IL-18 may not be the mediators of inflammation during CPV infection and opens up the
exciting possibility that CrmA functions to inhibit a novel target or activity.
CPVAIL-ipR Fails to Induce Inflammation on CAMs
IL-1 p is produced by a number of cell types and serves as proinflammatory
cytokine in response to cell injury (85). It has been demonstrated that the virally encoded
IL-ip receptor and not SPI-2/CrmA, is the molecule that is responsible for controlling the
actions of IL-1 p during vaccinia infections of mice (192). CPV also encodes a soluble
viral IL-1 P receptor that can bind IL-1 p and is thought to function as a decoy

172
Figure 32. Caspase-3 activity in the presence of chicken extracts. Extracts from
CPVAcrmA::lacZ confluent infections of CAMs harvested at 48 hours (70 pg) (-A-) or
10U of recombinant human caspase-3 (- -)or a mixture of extracts and purified enzyme
(- -) were assayed for the ability to cleave Ac-DEVD-AMC. Enzyme activity was
measured fluorometrically by the ability to cleave peptide substrate Ac-DEVD-AMC and
expressed as fluorescence signal units (FSU).

173
receptor regulating IL-1(3 action (381). If IL-ip were to be responsible for the
inflammation on the CAMs, then one would expect a recombinant CPV deleted for the
virus encoded IL-P receptor to also induce inflammation. In order to test this hypothesis,
CPV AIL-1 PR was constructed as outlined in Figure 33.
Briefly, plasmids containing homologous regions to the vIL-ipR were
constructed, interspersed with the selectable marker Eco.gpt. Using MPA selection
insertional vIL-1 PR mutant CPV were constructed. Since we did not have reagents to
test for loss of protein expression by immunoblot analysis, our examination of the
recombinant virus was limited to analyzing PCR products for loss of the gene. As
outlined in Materials and Methods (Recombinant Virus Construction), virus plaques
resistant to MPA were selected during the first round of plaque purification. Since this
plaque pick was likely to be contaminated with wild type CPV, additional rounds of
plaque purification were necessary. The CPVAIL-ipR virus was isolated following five
such rounds of plaque purification. As shown in Figure 33, using primers that bind vIL-
1 PR, PCR products were generated from viral DNA and resolved on agarose gels. The
gene encoding vIL-1 PR (present in wt CPV) is represented by a band migrating at 1 kbp
(Fig. 33B, lane 1) present in DNA samples made from the first round of plaque
purification. However, subsequent to plaque purification, vIL-1 PR did not get amplified
from recombinant CP VAIL-1 PR virus DNA. The 1.2 kbp PCR product band present in
Figure 33B, lane 1, represents the insertion of Eco.gpt into vIL-ipR gene in CPVAIL-
1 PR. As a control for viral DNA, CrmA was amplified using specific primers (data not
shown).

174
A.
CPV Genome
1
vIL-ipR
194007 194987
\/
224,501 bp
1 2
Figure 33. Construction of CPV AIL-1 PR. (A) The location of vIL-1 PR within the
CPV genome is indicated. The left and right flanking regions (hatched boxes) within
CPV vIL-ipR ORF were PCR amplified and cloned into pBluescript II KS+. The
E.coli.gpl gene was PCR amplified and cloned within CPV IL-1R left and right flanks
resulting in pBS-CPV-IL-IRKO. Insertional mutant virus CPV AIL-1 PR (KO) virus was
generated by homologous recombination and selection for MPA resistant (96) virus. (B)
PCR products were generated from CPVAIL-ipR viral DNA purified after 5 rounds of
plaque purification. A PCR product migrating at approximately 1.2 kbp represents the
recombinant KO viral construct (lane 1). A viral DNA sample prepared during the initial
stages of plaque purification show the presence of an additional 1.0 kbp PCR product
(lane 2) representing vIL-ipR in wt CPV that would be present along with the
recombinant virus in plaques, (see Recombinant Virus Construction in Materials and
Methods for detailed description of virus construct).

175
NBT
+ NBT
Figure 34. CPVAIL-lf3R fails to induce inflammation during CAM infections. Pock
formation by CPVAIL-ipR on the CAMs of 11 day old chicken embryos, 3 days post
infection. Membranes were untreated or stained with NBT to show the presence of
activated heterophils.

176
Using CPVAIL-ipR, 11 day old CAMs were infected and the lesions were examined 72
hours post infection (Fig. 34). The pocks caused by CPV AIL-1 PR were indistinguishable
from wt CPV pocks (compare Fig.21 and 34) which are hemorrhagic and non
inflammatory as they do not reduce NBT.
Taken together these results further add weight to the hypothesis that the
inflammation seen on CAMs in the absence of CrmA during CPV infections (Fig.21) is
not due to actions mediated by IL-1 p. Results from our study using recombinant CPV
infections are summarized in Table 7.

Table 7. Summary of results using recombinant CPVs.
CPV
CPVAcrmA
::lacZ
CPVAcrmA
::P35
CPVAcrmA
::SERP2
CPVCrmA
D303A
CPV AIL-1 PR
"Eco.gpt
LLCPK1 Infection
DAPI
No apoptosis
Apoptosis
No apoptosis
Apoptosis
Apoptosis
ND
Terminal caspase
-
+
-
+
+
ND
CAM Infections
Pock morphology
Red
White
White
White
White
Red
Heterophil influx
-
+
+
+
+
ND
MTT/NBT reduction
-
+
+
+
+
-
Virus Titers
106
o
1
o
106
106
104-105
ND
Terminal caspase
-
+
-
-
+
ND
chIL-18 processing
-
+
-
-
+
ND
chIL-l(5 processing
-
-
-
-
-
ND
hCaspase-1 inhibition
Inhibits
No inhibition
Inhibits
Inhibits
No inhibition
ND
ND-not determined

CHAPTER 4
DISCUSSION
Equivalency of SPI-2 and CrmA
CrmA was the first viral serpin to be characterized (316). All Orthopoxviruses
analyzed to date have been shown to contain the CrmA homolog SPI-2 (250). Overall
SPI-2 and CrmA share >90% identity and >92% similarity (250). Ectopic expression of
CrmA or SPI-2 can prevent apoptosis induced by TNF (407, 425). In addition both
CrmA and SPI-2 can inhibit caspase-1 with similar efficiencies (236). But despite their
high degrees of identity, published reports have shown that SPI-2 and CrmA may not
function similarly during virus infection. While CrmA can control inflammation in the
CAM model of infection, SPI-2 in vaccinia virus is unable to function in a similar manner
(304, 316). Previous reports have shown that during pig kidney cell infections, CrmA
functions to control apoptosis whereas RPV containing the functional CrmA homolog
SPI-2, is unable to prevent the induction of apoptosis (236). In the intranasal model of
infection, deletion of CrmA from CPV causes mild attenuation (one order of magnitude
in LD50 levels) whereas deleting SPI-2 from vaccinia virus has no effect on infection
(193,412).
In this study we attempted to determine the equivalency of the SPI-2 and CrmA
by switching the coding regions of the two genes between the two viruses RPV and CPV.
Swapping the entire ORFs between the two viruses allows us also to examine the effect,
if any, the individual viral genomes may have on the expression and function of SPI-
178

179
2/CrmA. SPI-2 expressed in CPV is observed to have similar kinetics of expression as
CrmA in wt CPV analyzed by immunoblots (Fig. 11). Similarly CrmA expressed in RPV
is observed to have comparable kinetics to SPI-2 from wt RPV (Fig. 10). To show
functionality of the two swapped gene constructs, LLC-PK1 cells were infected with
the recombinant RPV and CPV viruses. Here we show that RPVASPI-2::crmA is able to
completely block the induction of apoptotic morphology during infections of pig kidney
cells unlike in the case of wt RPV containing SPI-2 (Fig. 12). Cell extracts from
RPVASPI-2::crmA did not cleave DEVD-AMC, indicating the absence of caspase
activation (Fig. 14) unlike extracts from wt RPV infected cells. Similar experiments
performed with CPVAcrmA::SPI-2 virus show that the SPI-2 expressed in recombinant
CPV is able to block the induction of apoptosis in these cells similar to wt CPV (Figs 13
and 15).
Thus CrmA expressed in RPVASPI-2 protects infected swine cells from
undergoing apoptosis, whereas SPI-2 in wt RPV fails to function similarly. However, we
did notice that the level of apoptotic induction in wt RPV infected cells is much less and
stabilizes after 9 hours of infection, indicating that SPI-2 can partially reduce caspase
activation. In the context of RPV infections CrmA was clearly a better inhibitor of
caspase induction than was SPI-2. Whereas SPI-2 can function to completely block
apoptotic induction when expressed in CPV and can fully substitute for CrmAs function
during CPV infections of swine cells. These results also demonstrated that the minor
differences between CrmA and SPI-2 were inconsequential in CPV infections as both
proteins were able to function similarly in blocking the induction of caspase activity and
apoptosis. Therefore despite the high degree of homology between the two proteins

180
(>92%), their identical properties in vitro, the presence of similar but not identical
residues in the P5 and P6 positions RCL, the differences between the two molecules are
highlighted depending on the viral context of expression.
There are at least two major differing regions when comparing CrmA and SPI-2
molecules as seen in Figure 7. Overall, the CPV CrmA and RPV SPI-2 proteins are
>92% identical and within the RCL differ only at positions P5 and P6 (236). The second
region is within the viral serpin back-bone or region outside the RCL. Structural
comparison of CrmA with the prototypical serpin alpha-1 anti-trypsin reveals the absence
of a region termed helix D within CrmA (250). The helix D region is seen to function as
binding region for co-factors that modulate serpin function. Within CrmA, although
helix D is missing, it is replaced by the addition of a novel p-strand designated Sla
between residues 50 and 60. Interestingly RPV SPI-2 and CPV CrmA also differ in the
Sla region. It is therefore probable that differences seen in the function of CrmA and
SPI-2 are due to minor differences within the RCL and/or within the Sla region.
To differentiate between these two possibilities, it would be necessary to engineer
changes in SPI-2. First, the P5 and P6 residues in SPI-2 RCL could be mutated to mimic
CrmA RCL and see whether these changes have any effect on SPI-2 function within the
context of RPV infections. Second, the Sla region in SPI-2 could be made to look
exactly similar to the corresponding region in CrmA and the effect on SPI-2 function
within RPV context could be addressed. There lies the possibility that the differences
seen between SPI-2 and CrmA function within RPV are due to differences not associated
with either the RCL or Sla regions.

181
Of interest however, is the question of whether the differences seen between SPI-
2 and CrmA have any consequences for RPV or CPV infections in vivo. While there was
no significant difference between the LD50 of CPV and RPV in Balb/c mice, clearly there
are differences between the pathology caused by CPV and RPV infections (412). CPV
infection of mice was always accompanied by severe pulmonary hemorrhage as opposed
to RPV infections that did not caused pulmonary hemorrhage at any time during
infection. RPV infections caused viremia, which was absent from CPV infections.
RPVASPI-2 infected mice showed more extensive infiltrate of inflammatory cells in the
lungs of infected mice when compared with RPV infected mice indicating that SPI-2
played a role in limiting pulmonary inflammation. However, in the case of CPV
infections, CPVAcrmA showed decreased pulmonary pathology and inflammation
compared with CPV infected mice. It has been postulated that the pathology associated
with CPV infections is due to necrosis, which could explain the increased inflammatory
cell response as opposed to CPVAcrmA infections, which probably causes apoptosis in
infected cells, and therefore show decrease in pulmonary inflammation.
The availability of RPV and CPV expressing CrmA and SPI-2 respectively could
be used to study the effects of these genes on pulmonary pathology associated with virus
infection of mice.
Potential Applications
Our results highlight the fact that although the orthopoxviruses RPV and CPV are
considered to be very similar, they behave quite differently during infections of swine
cells and at least RPV is able to differentiate the minor differences between SPI-2 and

182
CrmA. If the differences between the properties of SPI-2 and CrmA as we have shown
were indeed dependent on the differences within the RCL (notably at P5 and P6), then it
would be of importance to consider these regions when designing therapeutic peptide
inhibitors of caspases (288). Differences as far as the P5 position have been shown to
influence substrate specificity (239). Peptide inhibitors of caspases are typically small
and comprise of residues meant to mimic RCL targeting of enzymes. Caspase-1
inhibitors have shown promise in the treatment of rheumatoid arthritis. Peptide inhibitors
designed for the inhibition of caspases involved in apoptosis are yet to reach the clinical
stage. Most of the work in therapeutics is centered on inhibiting caspase-3, the terminal
apoptotic caspase. In animal models of ischaemia, caspase inhibition improved survival.
Currently available peptide ketone inhibitors of caspases are relatively non-specific for
individual caspase members but do provide evidence for the therapeutic potential in
blocking caspase activity in certain disease models. While designing such molecules it
may be worth considering the pathological context of usage since our experiments show
that minor differences in the P5 and P6 regions of an RCL may be sufficient to modify
the outcome of an infection.
Therapeutic peptides based on targeting apoptotic and inflammatory caspases are
still in their infancy. Nevertheless this is an emerging and promising area of clinical
research as evidenced by various pharmaceutical companies that have an active interest in
the development of these technologies.

183
P35 Replacements of SPI-2 and CrmA
The goal of this study was to determine the functional capability of SPI-2/CrmA
during virus infection. If SPI-2/CrmA were simply functioning as caspase inhibitors then
replacing SPI-2 in RPV and CrmA in CPV with other caspase inhibitors such as P35
should produce recombinant virus infections of swine cells that prevented caspase
induction. To counter the argument of species specificity of using caspase inhibitors, we
chose to use P35, which has been shown to inhibit caspase induction in a number of
systems (250). However when we constructed recombinant virus RPVASPI-2::P35, we
encountered some unexpected results. P35 expressed in recombinant RPV begins to
disappear after 8 hours post infection of pig kidney cells as analyzed by immunoblots
(Fig. 16A). This unavailability of P35 at later times of infection explains the results we
observed with RPVASPI-2::P35 infections which failed to prevent apoptotic induction in
these cells judged both by DAPI stain and Ac-DEVD-AMC cleavage activity in cell
extracts (Figs. 12 and 14). However this phenomenon seems to be restricted to RPV
expressed protein.
P35 expressed in recombinant CPVAcrmA::P35 virus is stable throughout the
course of virus infection (up to 16 hours post infection) and was able to prevent the
induction of apoptosis in swine cells (Figs. 16B, 13 and 15). Thus while P35 was able to
replace CrmA function in CPV, the expression of P35 in RPV deleted for SPI-2 increased
the rate of Ac-DEVD-AMC cleavage in infected cell extracts when compared to extracts
from wt RPV infections that contain SPI-2. It was quite clear that P35 could not
substitute SPI-2 function in RPV. In the presence of AraC (inhibitor of viral DNA
synthesis), P35 expressed in RPV seems to be more stable indicating that the

184
phenomenon of P35 availability during RPV infections is related to events that occur
during/after viral DNA replication (Fig. 17). Interestingly the induction of caspase
activity also coincides with the timing of viral DNA replication and can be blocked by
AraC (data not shown). Whether P35 stability in RPV is related to caspase induction or
to some other late viral event is yet to be resolved.
It is possible that in RPV infections, P35 is processed/degraded by either a viral or
cellular protease that is activated after 8 hours of infection. Another possibility is that
P35 is inherently unstable but in CPV is rendered stable by another viral factor. We have
also noticed that at MOI of 15 or more, even within the context of CPV, P35 is unable to
completely block the induction of caspase activity in infections of pig cells (data not
shown). Therefore it seems probable that the stability of P35 may depend on events
associated with the virus infection, and this state is exacerbated in RPV but not CPV;
where P35 is able to function as a caspase inhibitor at MOI of 10. Since P35 expressed
normally in CPV is able to function as a caspase inhibitor during virus infection, we
continued to use this recombinant virus in our in vivo studies using the CAM model of
infection.
Function of CrmA during CPV Infections of CAMs
Previous reports of infected CAMs have dealt with lesion morphology and
infiltrating cell types. In this study using the CAM model of infection, we investigate the
biochemical properties of CrmA in order to gain a better understanding of its role during
CPV infection. Consistent with the ability of CrmA to inhibit the inflammatory response
during CPV infections of CAMs, it has been shown that CrmA can directly inhibit

185
caspase-1 in vitro (331). Caspase-1 processes proIL-ip and proIL-18 to the mature pro-
inflammatory cytokines (85). CrmA as mentioned earlier can also inhibit the induction of
apoptosis by ectopic expression (93, 117, 407) and during CPV infection of pig kidney
cells (236, 332). These observations correlate with studies showing that CrmA inhibits
the apical caspases -8, -9 and -10 (118). Thus CrmA regulates both inflammation and
apoptosis during CPV infection. Results from our study are summarized in Table 7.
Requirement of Intact RCL for CrmA Function In Vivo
In order to determine if there are other regions within CrmA that may contribute
to its function during CPV infection, we mutated the PI Asp residue in CrmA RCL to
Ala. Mutation at the PI position of a serpin alters target specificity (368). Infections of
CAMs with CPVCrmA D303A recombinant virus produced white, inflammatory lesions
(Fig. 21). The CrmA D303A mutant pocks had essentially the same characteristics as
those from the null mutant infections with respect to inflammatory cell influx (Fig. 22),
virus yields (Fig. 24), caspase induction (Fig. 26) and the ability of pock extracts to
process proIL-ip and proIL-18 (Figs. 27-29). Therefore, we have shown for the first
time that CrmA functions by a protease inhibition mechanism in vivo and that serpin
function is required in order to inhibit inflammation and apoptosis on CAMs.
Regulation of Inflammation and Apoptosis by CrmA are Distinct Functions
We replaced the CrmA gene within CPV with other caspase inhibitors to
determine if CrmA functions solely by inhibiting caspases. If the preceding hypothesis
were true, P35 and SERP2 replacements of CrmA in CPV should behave like wild type

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CPV during infections of CAMs. We did not expect both P35 and SERP2 recombinant
viruses to produce white, inflammatory pocks containing activated heterophils (Figs. 21,
22 and 23). However, P35 and SERP2 proteins function in the context of CPV to restore
virus yields within pocks to wild type levels unlike in the case of the null mutant or the
CrmA D303A mutant viruses (Fig. 24). The restoration of virus yields in the case of both
P35 and SERP2 recombinant virus infections means that inflammation does not
necessarily correlate with reduction in pock titers. P35, SERP2 and CrmA block the
induction of apoptosis on CAMs, whereas pocks from the CrmA null mutant and CrmA
D303A mutant virus infections induced a caspase-3-like activity as seen by the ability to
cleave Ac-DEVD-AMC (Fig. 26). These results correlate with virus yields from pocks
(Fig. 24) confirming our suspicion that reduction in virus yields was a result of failure to
control apoptosis and not due to an inflammatory response. This also opened up the
possibility that the regulation of inflammation and the regulation of apoptosis by CrmA
were two distinct functions that may not be dependent on caspase inhibition alone. Thus
P35 and SERP2 replacements of CrmA in CPV are able to function as inhibitors of
caspase activation but do not inhibit the induction of inflammation within pocks.
Inflammation During CPV Infection may not be due to IL-lp or IL-18
It has been assumed that the lack of inflammation within wt CPV pocks is due to
the ability of CrmA to inhibit the proinflammatory caspase-1 (304) which can cleave
proIL-ip and/or proIL-18 which would lead to formation of the proinflammatory
cytokines IL-1 (3 and IL-18 (16). However, the data presented here as well as some
published data argue against this hypothesis.

187
To address the issue of whether the inflammation seen on the CAMs was indeed
dependent on and mediated by caspase-1, we assayed extracts for the ability to process
the two capase-1 substrates proIL-18 and proIL-1P (Figs. 27, 28 and 29). Caspase-1, like
all other caspases specifically cleaves substrates after certain Asp residues. Unlike the
mammalian system where maturation of IL-ip requires caspase-1 to process the inactive
precursor initially at Asp27 (site 1) followed by cleavage at Aspl 16 (site 2) (395), the
chicken proIL-ip sequence does not contain either Asp27 or Aspl 16 residue required for
cleavage by caspase-1 that would release the mature cytokine (446). The mechanism of
IL-1P maturation in chickens is currently unknown. The chicken proIL-1 P sequence is
similar to proIL-ip sequences from frog and fish, which also do not contain the
conserved Asp sites found in mammals (475). Biological activity has been shown with a
putative chicken IL-ip recombinant protein (446). While chicken caspase-1 shares 44%
homology to mammalian caspase-1 (178), there is no functional data to show processing
of either chicken proIL-18 or proIL-1 P by chicken caspase-1.
Indeed, the failure of recombinant human caspase-1 to process chicken proIL-ip
(Fig. 27) is also consistent with the idea that the mechanism of avian IL-1 p activation
may not be mediated by caspase-1. If the mechanism of processing chicken proIL-ip
involved an alternate non-caspase-1, then one would expect the inflammatory extracts of
CPVAcrmAdacZ, CPVCrmA D303A, CPVAcrmA::P35 and CPVAcrmA::SERP2 to
process the chicken proIL-1 p precursor. We found no evidence for chicken proIL-1 p
processing from any infected extract (Fig. 27). Since we were assaying for chicken
caspase-1 activity, we chose to use an alternate caspase-1 substrate namely mouse proIL-
1P which contains the conserved Asp residues required for processing by caspase-1. We

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failed to detect mature IL-1 (3 under any CAM infection condition and also found that P35
and SERP2 function like CrmA in blocking caspase-1 (when added exogenously)
mediated processing of IL-ip (Fig. 28). This suggested that during CPV infection if
caspase-1 were to be involved, P35, SERP2 and CrmA could block it.
These results suggested that chicken IL-1 (3 is not involved in the inflammation
during CPV infection of CAMs. Alternatively if chicken IL-1P is responsible for the
inflammation on CAMs, chicken proIL-1P is possibly active as a precursor. We would
not be able to test pocks for proIL-1 p production since all the inflammatory pocks
contained high levels of heterophils, which are known producers of IL-ip.
Because of the ambiguity surrounding activation of avian IL-1 p, we chose to
investigate the ability of infected CAMs to instead cleave proIL-18 to active IL-18 as an
indicator of caspase-1 activity. The chicken proIL-18 sequence contains the conserved
Asp29 residue required for caspase-1 mediated cleavage (354). Therefore it was not
surprising that CPVAcrmA::lacZ and CPVCrmA D303A extracts from CAMs that were
known to contain caspase activity (Fig. 26), were able to process chicken proIL-18 (Fig.
29). Despite reports of caspase-3 being able to process proIL-18 (122, 133), we found
little, if any, processing of chicken proIL-18 by up to 15 U of recombinant human
caspase-3 (Fig. 30). The inability of human caspase-3 to process chicken proIL-18 could
be due to species specificity, i.e. a low affinity of human caspase-3 for the chicken
substrate. However, we have also shown that the chicken proIL-18 processing ability by
CPVAcrmA::lacZ infected CAM extracts could be inhibited by pre-incubating extracts
with Ac-DEVD-CHO, a caspase-3 inhibitor or Z-VAD-FMK, a pan-caspase inhibitor but
not Ac-WEHD-CHO, a caspase-1 inhibitor (Fig. 31). Therefore we conclude that the

189
proIL-18 processing activity observed in CPVAcrmA::lacZ extracts was due to a caspase-
3like activity and not due to caspase-1.
IL-1 p is produced by a number of cell types during tissue injury (85) and is
controlled in two ways during orthopoxvirus infection. In addition to regulating IL-1 P at
the post-translational level by blocking caspase-1 using CrmA, orthopoxviruses also
encode a soluble IL-1 P receptor (vIL-1 PR). It has been shown in the intranasal mouse
model of infection that vIL-1 PR rather than SPI-2 (CrmA homologue in vaccinia virus)
controls the febrile response mediated by IL-1 P (192). In order to verify this observation,
we deleted the vIL-1 PR in CPV and the resulting virus was used to infect CAMs (Fig.
34). If IL-1 P were to be involved in the inflammation on the CAMs, one would expect
CPV AIL-1 pR infections to produce white, inflammatory pocks. We constructed an
insertional mutant CPVAIL-ipR and used this virus to infect 11 day old CAMs.
CPV AIL-1 PR virus produced red, hemorrhagic, non-inflammatory pocks like wild type
CPV, which did not reduce NBT (Fig. 34). This observation strengthened our hypothesis
that the inflammation on CAMs was not mediated by IL-1 p. Since the inflammatory
response on CAMs is mediated by heterophils during CPVAcrmA::lacZ infection, it is
probable that heterophil chemoattractants other than IL-1 P are involved. The potential
candidates would include leukotrienes, TNFa, IL-8, IL-6 and to a lesser extent IL-12
since this requires a functional cell mediated response, which is absent in 10 day old
chick embryos.

190
Models of Inflammatory Response on CAMs
We propose three possibilities to explain the inflammatory response on CAMs.
First is the possibility that our observations are due to species specificity. In the case of
SEPR2 replacements of CrmA, this would not be surprising. We have observed that
during infections of pig kidney cells, SERP2 was unable to inhibit the activation of
caspases (Fig. 20). However, SERP2 was able to inhibit caspase activation during
infections of chicken cells (Figs. 25 and 26). Therefore it is apparent that species
specificity could play a role in the function of SERP2. On the other hand P35 functioned
consistently as an inhibitor of apoptotic induction and caspase activation in both the pig
and chicken systems (Figs. 13, 15, 25 and 26), an observation consistent with reported
properties of P35 functioning in heterologous systems (250). Since neither P35 nor
SERP2 were able to prevent inflammation within the context of CPV infections of
CAMs; there does exist the possibility of species specificity of chicken caspases to P35
and SERP2 (assuming that CAM inflammation is caspase mediated).
Second, there is some evidence in literature for a possible role played by CrmA in
arachidonate metabolism during virus infection. It has been shown that orthopoxviruses
such as CPV require arachidonate metabolites during replication in vivo and in vitro
(303). Blocking the lipoxygenase pathway (but not the cycloxygenase pathway)
prevented pock formation on CAMs and impaired virus replication in tissue culture.
While infection of chicken embryo fibroblasts with CPV up regulated 15-lipoxygenase
and 12-lipoxygenase product formation, deletion of CrmA in CPV caused a dramatic
increase in the formation of products similar to 14(R),15(S) diHETE
(dihydroxyeicosatetraenoic acid), 15HETE and 12HETE (305). Since poxviruses have

191
an absolute requirement for the lipoxygenase pathway for replication, there is a potential
to form mediators of inflammation such as leukotrienes, which are derived from the
lipoxygenase pathway. Lipoxygenase metabolites have been shown to be both positive
and negative regulatory effectors of the immune system (323, 326, 400, 401). 12(S)-
HETE enhances tumor cell adhesion to endothelium by up regulating the expression of
integrins (401). 12(S)- and 15(S)-HETE are known to be involved indirectly in rat
carcinoma cell survival (400). Interestingly 14(R), 15(S) diHETE has been shown to
inhibit leukotriene B4 function in neutrophils (323) and natural killer cell activity (326).
It is unclear at present if any of the lipoxygenase metabolites are directly involved in the
induction of inflammation on the CAMs during CPV infection or if they serve as
intermediates in pathways that lead to the production of inflammatory mediators. While
the accumulation of diHETE during CPV and CPVAcrmA infections of CAMs was
detected as early as 1 hour post infection (305), caspase activity due to CPVAcrmA can
only be found after 8 hours of infection in tissue culture (424). Our data supports a
model of dual targets for CrmA, where the regulation of apoptosis is brought about by its
direct interaction with caspases and the regulation of inflammation is brought about by
inhibiting the formation of inflammatory mediators which may include or induce
formation of lipoxygenase metabolites. Thus using other caspase inhibitors such as P35
and SERP2, we were able to replace the anti-apoptotic function of CrmA, but were
unable to inhibit the inflammatory response on CAMs.
There is some evidence to support the dual function hypothesis. It has been
reported that SERP2 in wt myxoma virus infections, functions to prevent the induction of
apoptosis of rabbit cells (257). Similarly CrmA expressed in recombinant myxoma virus

192
is able to function like SERP2 to inhibit apoptotic induction in rabbit cells (Peter Turner
and Richard Moyer unpublished). SERP2 was also shown to be a much weaker inhibitor
of caspase-1 and granzyme B than CrmA (424). Studies involving infections of
European rabbits reveal a role for SERP2 in myxoma virus virulence (257). When
SERP2 is replaced by CrmA in myxoma virus, the disease is markedly attenuated
although 60% of the rabbits do succumb to the infection (Amy MacNeill and Richard
Moyer unpublished). Interestingly the primary lesions of rabbits infected with
MYXASERP2::crmA look like those of the SERP2 deletion mutant. In addition there is
some reduced ability of recombinant myxoma expressing CrmA to cause spread and
secondary lesion development. Since CrmA is unable to completely restore SERP2
function in myxoma virus, it suggests that the function of SERP2 is more than just
inhibiting caspases. The natural target of SERP2 during myxoma virus infections is yet
to be elucidated.
The third possible reason for the inflammation on CAMs is that the chicken
precursor proIL-lp itself is biologically active. In the mammalian system the biological
activity of IL-1 is shared by IL-la and IL-1 P (84). A third member of the family is the
IL-1 receptor antagonist (IL-IRa), which blocks the activities of IL-la and IL-ip by
competing for the receptors (86). ProIL-ip is biologically inactive, whereas proIL-la is
fully functional as a precursor (82). In chickens, the homologs of IL-la or IL-IRa are
yet to be found (384). Since the IL-1 system in chickens is more closely related to that of
amphibians and fish than to that of mammals (475), it is conceivable that IL-la is not
present in chickens and thus the function of IL-1 would be mediated by IL-1 p alone.

193
Furthermore, this would suggest a novel mechanism for pro-inflammatory IL-ip
induction that can be blocked by CrmA but is independent of its anti-caspase function.
Genetic space is limited for infectious agents such as poxviruses. It is therefore
not surprising that some of the poxviral serpins are bi-functional. Although the natural
target for the poxviral serpin SPI-3 of CPV is not known, studies have shown that SPI-3
functions as a serpin in vitro (420) and also controls cell fusion in a serpin independent
manner (423). Similarly CrmA could be functioning to inhibit the induction of apoptosis
by its direct interaction with caspases and could be regulating inflammation by a yet
unknown target or mechanism.
Future Studies
CrmA definitely has a role during CPV infection. Its precise function during
virus infection is not yet known and the natural target for CrmA is yet to be elucidated.
Owing to the limited availability of reagents for chickens, it will be useful to use the
mouse models of infection to answer some of these questions. A number of mouse
models of infection have been discussed that measure inflammation. Not all of these
have been tested for the effect of the CrmA gene during virus infection. As mentioned
earlier, deletion of CrmA in the intranasal model shows only mild attenuation while
deletion of SPI-2 has no effect on infection (193, 412). Most recently mice infected via
the intradermal route in the ear, show an increase in lesion pathology due to the absence
of SPI-2 (418). Studies from our lab show promise for the use of an intratracheal route
during CPV infection (Amy MacNeill and Richard Moyer unpublished). Furthermore a
number of knockout mice for the different components of the IL-1 (Table 1) and IL-18

194
system are now available which could answer questions regarding the involvement of
CrmA in one or more of these cytokine pathways. The possibility of using knockout
mice in these studies is very promising.
The question of whether CrmA functions as a caspase inhibitor to modulate the
inflammatory response on CAMs could also be verified by use of cell permeable peptide
inhibitor of caspases in this model system. Z-VAD-FMK, a pan-caspase, cell permeable,
inhibitor can be used to pre-treat membranes prior to the inoculation of CPVAcrmA::lacZ
virus. If the generation of white inflammatory pock phenotype by the CrmA null mutant
virus were mediated by caspase activation, the presence of Z-VAD-FMK would be
expected to block caspase activity during virus infection and prevent the generation of an
inflammatory response. However, the generation of white, inflammatory pocks by
CPVAcrmA::lacZ, in the presence of functional pan-caspase inhibitor Z-VAD-FMK,
would be indicative that inflammation during CPVAcrmA::lacZ infections is not caspase
mediated and furthermore indicate that CrmA functions to inhibit a novel pathway in the
CAM system.
Orthopoxviruses encode a soluble IL-18 binding protein (292). Since the
possibility exists that IL-18 mediates the inflammation on CAMs, it would be interesting
to examine the contribution of viral IL-18 binding protein in the CAM model. A mutant
CPV lacking viral IL-18 binding protein can be constructed and used to infect CAMs.
There is no evidence to date, suggesting a role for CrmA in interfering with the
generation of proinflammatory mediators such as IL-1P or TNF-a. Recent advances in
understanding innate immunity have shown a role for TLR involvement in inflammation
(172, 293). TLR4 has been shown to mediate host response to LPS and hence Gram-

195
negative bacteria (172). TLR2 recognizes bacterial lipoproteins from a number of Gram
positive bacteria (172). It is possible that CrmA interferes with the generation of ligands
that could trigger TLR mediated innate response. CPV contains homologs to A46R
(ORF VI76 in CPV) and A52R (ORF VI82 in CPV), two proteins found in vaccinia
virus that has been shown to antagonize IL-1, TLR and IL-18 signaling (172,
http:://www.poxvirus.org). If TLRs play a role in mediating inflammation during CPV
infection of the CAM, then it is possible that VI76 or VI82 gene products may also be
involved in blocking this host immune response. A mutant CPV lacking either VI76 or
V182 could be expected to induce an inflammatory reaction during CAM infection and
thereby produce white pock phenotype.
We were unable to detect caspase-1 activity under any infection condition.
However, a number of alternate proIL-ip and proIL-18 processing mechanisms have
been discussed earlier. There exists a possibility that CrmA could be involved in blocking
the activity of a non-caspase enzyme that processes precursor IL-1 p or IL-18. Neutrophil
enzyme PR-3, has recently been shown to generate IL-18 (99, 389), it is yet to be seen if
CrmA can interfere with PR-3 activity. Similarly, a number of matrix metalloproteases
(MMP) have been shown to process proIL-1 P (355). Indeed homologs of some of these
MMPs have been identified in chickens (64, 137, 328). The ability of CrmA to inhibit
avian MMP activity can be tested.
It remains a possibility that during the course of future studies novel targets for
CrmA interaction will be discovered.

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BIOGRAPHICAL SKETCH
Rajkumar Nathaniel was bom on April 13, 1971 in Madras, India. He and his
brother Vijaykumar grew up in Mysore, India. He completed the Bachelor of Science
degree from St. Josephs College at Bangalore in 1991. After his graduation he was
trained as a Medical Microbiologist at the Christian Medical College, Vellore, India.
From 1993 to 1994 he worked as a Research Assistant at AstraZeneca Research,
Bangalore. In the Fall of 1994, he joined the graduate program at the Department of
Microbiology, University of Central Florida, Orlando, where he obtained the Master of
Science degree. In 1996 he joined the Interdisciplinary Program in Biomedical Sciences
at the University of Florida. He entered Dr. Richard Moyers laboratory in the
Department of Molecular Genetics and Microbiology in the Summer of 1997 to begin
work on his dissertation. He married Mamatha Chandrakumar in 1999. Following the
completion of his Ph.D., he joined the laboratory of Dr. Richard Benya at the University
of Illinois at Chicago to begin postdoctoral training.
239

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Richard W. Moyer, C
Professor of Molecul
Microbiology
enetics and
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Molecular Genetics and
Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Lung-Ji Chang
Associate Professor of Molecular
Genetics and Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
WMl
Michael'T/Clare-Saflzi'er
Associate ProfessorVDPathology,
Immunology, and Laboratory
Medicine

This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
May 2003



108
Table 6. List of constructed recombinant viruses
Recombinant Viruses Constructed
Virus and plasmids used
1. RPVASPI-2::Eco.gpt
RPV and pBS-Rgpt
2. RPVASPI-2::CrmA
RPVASPI-2::Eco.gpt and pRSG
3. RPVASPI-2::lacZ
RPV and pRglacZ
4. RPVASPI-2::P35
RPVASPI-2::lacZ and pR35
5. CPVAcrmA::Eco.gpt
CPV and pBS-Cgpt
6. CPVAcrmA:SPI-2
CPVAcrmA: :Eco.gpt and pCSG
7. CPVAcrmA::lacZ
CPV and pCglacZ
8. CPVAcrmA::P35
CPVAcrmA::lacZ and pC35
9. CPVAcrmA::SERP2
CPV AcrmA: :lacZ and pCSERP2
lO.CPVCrmA D303A
CPV AcrmA: :lacZ and pBS-D303ACgS/A
ll.CPVAIL-lpR::Eco.gpt
CPV and pBS-CPVIL-IRKO


37
MC148R molecule is able to bind receptors but does not transmit signal as it lacks a
region critical for receptor activation (78, 206).
The second group of viral proteins is chemokine receptor homologs. Virus
encoded chemokine receptor homologs are typically cell membrane bound and function
to bind chemokines. These include the ORF74 protein found in a number of herpes
viruses that binds CXC chemokines (342). The US28 gene of cytomegalovirus is a
functional CC chemokine receptor (36). In addition this virus also encodes US27, UL33
and UL78 proteins with homology to CC chemokine receptors some of which have been
found to localize on viral membranes (109, 240, 330). Swinepox virus encodes the K2R
molecule that is potential receptor of CXC chemokines (246) (Traci Ness and Richard
Moyer unpublished). Capripoxvirus also contains a putative CC chemokine receptor the
Q2/3L ORF (54). Molluscum encodes MC148, a MIP-ip homolog that was shown to
antagonize the chemotactic activity of a number of CC and CXC chemokines on different
cell types (78).
The third mechanism employed by viruses is to secrete binding proteins that
sequester chemokines and prevent chemotaxis of inflammatory cells to the site of
infection. Examples of these would include the myxomavirus M-T7 protein that was
originally discovered as an IFNy receptor but has subsequently been found to also bind C,
CC and CXC chemokines (214, 429). The M-Tl gene product of myxomavirus and
rabbitpoxvirus secreted 35 kDa proteins function similarly in binding CC chemokines
with high affinity and IL-8 (CXC) with a lower affinity but share limited homology (129,
216). In vivo studies indicate that M-Tl deletions in myxoma do not affect virulence but
the absence of M-Tl increased infiltrating monocytes and macrophages at the site of


31
frog and fish, which also do not contain the conserved Asp sites found in mammals (475).
Matrix metalloproteinases (MMPs) have also been implicated in the processing of
mammalian proIL-ip (355). Gelatinase A (MMP-2) and B (MMP-9) are among the
MMPs shown to cleave proIL-1 (3. An avian gelatinase that shares homology with MMP-
2 is observed to be over expressed in chicken embryo fibroblasts transformed with Rous
sarcoma virus (64). In addition, a chicken enzyme similar to MMP-9 has been cloned
from macrophages that differ from its mammalian counterpart in sequence and
biochemical function (137). In light of alternative mechanisms of processing observed
for mammalian IL-ip, it is worth noting that chicken heterophils treated with LPS,
zymosan or phorbol myristate acetate (PMA) show increased MMP production (328).
Although there seem to avian homologs to mammalian enzymes that have been
implicated in IL-1P maturation, there is no experimental data to prove that any of the
avian enzymes actually function to process avian IL-1 p.
The chicken IL-1RI was cloned from chicken embryo fibroblasts (136).
Interestingly the cytoplasmic region of chicken IL-1RI is nearly 80 % homologous to
both its mammalian counterpart as well as the Drosophila Toll receptor. Like other TLRs
the chicken receptor also has an extracellular Ig-like region although sequence
divergence is higher in this region. Soluble chicken IL-1RI was shown to inhibit IL-1
like activity released from LPS induced macrophages confirming an antagonistic role for
this molecule (196). The chicken homologs of IL-1 a, IL-1RII or IL-1 Receptor
antagonists are yet to be identified.


Table 1 Characterization of mice deficient for different components of the EL-1 system
IU1-specific components
Components shared with IL-18
In vitro or
in vivo
Receptors
Agonist ligands
Antagonist ligands
Signalling
Processing enzyme
response to
IU1RI
IL-lRAcP
EL-lot
ID10
IDIRa
IRAK
caspase-1
LPS
Normal
ND
ND
Variable
Increased
Reduced
Reduced
Turpentine
Reduced
ND
Normal
Reduced
Increased
ND
Normal
idi
Absent
Absent
ND
Increased
ND
Reduced
ND
EL-18
ND
ND
ND
ND
ND
Reduced
ND
IJsteria
Increased
Normal
Normal
Normal
Reduced
Normal
ND
monocytogenes
susceptibility
susceptibility
susceptibility
susceptibility
susceptibility
susceptibility
Ischaemia
Reduced
ND
ND
ND
ND
ND
Reduced
/reperfusion
Allergen
Reduced
ND
ND
ND
ND
ND
ND
challenge
Pancreatitis
Reduced
ND
ND
ND
ND
ND
Reduced
Spontaneous
inflammation
No
No
No
No
Arthropathy
arteritis
No
No
From reference 98


194
system are now available which could answer questions regarding the involvement of
CrmA in one or more of these cytokine pathways. The possibility of using knockout
mice in these studies is very promising.
The question of whether CrmA functions as a caspase inhibitor to modulate the
inflammatory response on CAMs could also be verified by use of cell permeable peptide
inhibitor of caspases in this model system. Z-VAD-FMK, a pan-caspase, cell permeable,
inhibitor can be used to pre-treat membranes prior to the inoculation of CPVAcrmA::lacZ
virus. If the generation of white inflammatory pock phenotype by the CrmA null mutant
virus were mediated by caspase activation, the presence of Z-VAD-FMK would be
expected to block caspase activity during virus infection and prevent the generation of an
inflammatory response. However, the generation of white, inflammatory pocks by
CPVAcrmA::lacZ, in the presence of functional pan-caspase inhibitor Z-VAD-FMK,
would be indicative that inflammation during CPVAcrmA::lacZ infections is not caspase
mediated and furthermore indicate that CrmA functions to inhibit a novel pathway in the
CAM system.
Orthopoxviruses encode a soluble IL-18 binding protein (292). Since the
possibility exists that IL-18 mediates the inflammation on CAMs, it would be interesting
to examine the contribution of viral IL-18 binding protein in the CAM model. A mutant
CPV lacking viral IL-18 binding protein can be constructed and used to infect CAMs.
There is no evidence to date, suggesting a role for CrmA in interfering with the
generation of proinflammatory mediators such as IL-1P or TNF-a. Recent advances in
understanding innate immunity have shown a role for TLR involvement in inflammation
(172, 293). TLR4 has been shown to mediate host response to LPS and hence Gram-


141
Infections of LLC-PK1 Cells with CPV Recombinants: Ac-DEVD-AMC Cleavage
Activity
To confirm biochemically, the morphological indications of apoptosis (Fig. 19),
we analyzed infected pig kidney cell extracts for the ability to cleave Ac-DEVD-AMC
(Fig. 20). Cleavage of Ac-DEVD-AMC is indicative of terminal caspase activity present
in cells undergoing apoptosis. Extracts were prepared at 12 hours post infection and
incubated in the presence of Ac-DEVD-AMC. Rates of Ac-DEVD-AMC cleavage were
calculated and are shown in Figure 20. As expected CPV infected extracts had little or
no peptide cleavage activity. CPVAcrmA::P35 extracts behaved like wt CPV extracts
and failed to cleave Ac-DEVD-AMC similar to results obtained in previous experiments
(Fig. 15).
However, CPVAcrmA::SERP2 and CPVCrmA D303A extracts behaved similarly
to CPVAcrmA::lacZ extracts and showed significant cleavage of the fluorogenic peptide
(Figure 20). These results correlate with DAPI stain results obtained in Figure 19 and
confirm the presence of caspase activity in CPVAcrmA::lacZ, CPVAcrmA::SERP2 and
CPVCrmA D303A infected cell extracts. Thus SERP2 is unable to replace CrmA to
prevent the induction of caspases during CPV infections of swine cells. Perhaps this is
due to species specificity of SERP2, i.e. a poor affinity of SERP2 for pig caspases (but
not rabbit caspases). Indeed SERP2 has been reported to function as an apoptotic
inhibitor during myxoma virus infections of the European rabbit (257). SERP2 has also
been shown to prevent apoptotic induction during myxoma infections of rabbit kidney
cells (Peter Turner and Richard Moyer, unpublished). Our results with CrmA D303A
mutant virus further confirm the importance of the RCL in CrmA function and also


205
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121
A.
CPV Genome
1
CrmA
192,309 193,334
\/
224,501 bp
GM 17
CrmA ORF
GM 21
GM 19
GM 22
B.
Xbal
LacZ
Hindi II
Xbal
P35
Ncol
SERP2
Smal
Ncol
CrmA D303A
Smal
CrraA Left Rank
Apal (2708)
Figure 9. Construction of recombinant CPV. (A) The location of the CrmA gene
within the virus chromosome is indicated. The flanks upstream and down stream
(hatched boxes) of the CrmA coding region were amplified using the primers indicated.
(B) Illustration of the shuttle vector used to replace CrmA in CPV with either lacZ, P35,
SERP2 or CrmA D303A. The various genes were cloned into pBS-CgS/A containing the
E.coli gpt gene outside the CrmA flanks (hatched boxes), which were then used to
generate recombinant CPVs by transient dominant selection (97). Restriction sites used
for cloning are indicated. See Materials and Methods (Recombinant Virus Construction)
for each individual virus construct.


LIST OF TABLES
Table page
1 Characterization of mice deficient for different components of the IL-1 system 15
2 List of mice deficient in the IL-1 system 16
3 Taxonomy of poxviruses 66
4 List of Primers 90
5 List of plasmids constructed 107
6 List of constructed recombinant viruses 108
7 Summary of results using recombinant CPVs 177
viii


206
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71
and 10 divergent orphans (368). Although most serpins function to inhibit serine
proteases, there exist examples of cross class inhibition such as that exhibited by the
poxviral serpin CrmA and the cellular PI-9 which can inhibit both serine and cysteine
proteases (e.g. caspases). In addition some serpins such as ovalbumin are non-inhibitory
while corticosteroid binding globulin (CBG) and thyroxine binding globulin (TBG)
function as transport proteins (165, 311). In mammals serpins are involved in regulating
a number of processes including coagulation, fibrinolysis, inflammation, complement
activation, organ development and extracellular homeostasis as shown in Figure 4.
A metastable structural conformation is extremely important for the function of inhibitory
serpins. The conserved secondary structure is composed of three (3-sheets and at least 7
(typically 9) a-helicies. The exposed reactive center loop (RCL) is a region spanning
about 20 amino acids located at the C-terminus of the molecule which serves as the
proteinase recognition site as shown in Figure 5. Within the RCL lies the PI residue that
determines target proteinase specificity. The Pl-Pl peptide bond is cleaved during
scissile bond formation with the proteinase. Initially the proteinase forms a non-covalent
Michaelis-like complex by interactions involving residues flanking the scissile bond.
Attack by the active site serine (in the case of serine proteases) on the PI residue
leads to the formation of a covalent bond between the carbonyl of the PI residue and the
protease. At this point, the RCL begins to insert itself into (3-sheet A of the serpin while
still attached to the protease. The consequence of complete loop insertion is a 70A
translocation of the target proteinase that effectively distorts its active site resulting in
inactivation of the enzyme. The example of trypsin inactivation by alpha-1 antitrypsin is
shown in Figure 6. The formed acyl intermediate is stable from hours to weeks and is


237
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ACKNOWLEDGMENTS
A number of people have contributed to the completion of this work. I would like
to thank my mentor Richard Moyer for allowing me to start this project and also for
helping me complete it. Dick has been extremely patient and forgiving with me these
past 6 and some years. He continuously made efforts to train me as a scientist. Dick
helped me appreciate poxviruses. The members of my committee Lung-Ji Chang, Paul
Gulig, and Michael Clare-Salzler have also been instrumental in guiding me through
these years. They have sat patiently through many more committee meetings than they
would have wanted to. I would also like to thank Dr. Richard Condit and Dr. Sue Moyer
for valuable input and suggestions during joint lab-meetings.
The members of the Moyer Lab need special mention. They have been good
friends and work colleagues. Mike Duke taught me the difference between Republicans
and Democrats. Mike and I have had many philosophical discussions about the world we
live in and I will miss those interactions. Alison Bawden, Traci Ness and Kristin Moon of
the so-called Estrogen Sanctuary made sure I wasnt naive by the time they left the lab.
Ben Luttge and Lauren Brum have been wonderful work mates and strongly encouraged
my graduation process. Peter Turner has been generous with his time and was always
there when I needed technical advice. I am grateful to Marie Becker, for reading my
manuscripts and for her mothering us in the lab. Qianjun Li has been a good friend, work
neighbor and advisor.
11


137
infection as seen in CPVAcrmA::P35. We conclude that the issue of P35 stability is
probably due to different biochemical events that occur during RPV but not CPV
infections.
P35 expression from within CPVAcrmA::P35 is stable and the recombinant
protein is capable of substituting for CrmA function in preventing caspase induction.
Therefore we chose to concentrate our efforts in using this recombinant virus to further
elucidate the in vivo role of CrmA during CPV infections of CAMs.
Recombinant CPV Viruses Expressing SERP2 or CrmA D303A
CPV infections of 11 day old CAMs produce red, hemorrhagic, non-inflammatory
pocks whereas deletion of the CrmA gene from CPV induces white, inflammatory pocks
with lower virus yields (306). Therefore CrmA functions to increase virus yields and
inhibit the induction of an inflammatory response on CAMs. Using the CAM model of
CPV infection, our experiments were directed to address two general questions. The first
was whether CrmA inhibits inflammation on CAMs by functioning as a protease
inhibitor. The second was whether the ability of CrmA to inhibit caspases (in particular
caspase-1) was sufficient to explain the ability of CrmA to control inflammation and
virus yield during CAM infections. We first produced two additional recombinant CPVs
in addition to CPVAcrmA::P35 in which either the critical PlAsp, key to caspase
recognition, was mutated (CPVCrmA D303A) or the entire CrmA gene was replaced by
the SERP2 of myxoma virus. Both P35 and SERP2 have been described as potent
inhibitors of caspases (314, 474). The general strategy for construction of these viruses is


216
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140
prevent the induction of apoptosis judged both by DAPI staining of infected cells and by
testing cell extracts for caspase activity (Fig. 13 and 15). Similarly, pig cells infected
with CPVAcrmA::SERP2 or CPVCrmA D303A were stained with DAPI, the results of
which are shown in Figure 19. The presence of densely staining apoptotic nuclei
indicated that CPVACrmA::SERP2 infections looked similar to CPVAcrmA::lacZ
infected cells (Fig. 19). This result indicates that SERP2 failed to inhibit the induction of
apoptosis during CPVAcrmA::SERP2 infections and that SERP2 was unable to substitute
for CrmA function in CPV, confirming previous reports (424). Thus SERP2 despite
being a caspase inhibitor, fails to block apoptotic induction during virus infection of
swine cells.
CPVCrmA D303A virus infections of pig kidney cells likewise were
indistinguishable from those of CPVAcrmA::lacZ (Fig. 19). The D303A RCL mutant
virus infected cells showed morphological signs of undergoing apoptosis such as the
presence of apoptotic bodies. This result indicates that CrmA functions as a protease
inhibitor during CPV infections to prevent the induction of apoptosis. The results
obtained from the D303A mutant virus underscores the importance of the PI position for
CrmA in the recognition of protease targets to prevent apoptotic induction. Since both
CPVAcrmA::SERP2 and CPVCrmA D303A viruses induce apoptosis in swine cells, we
expected to see caspase activity in extracts made from these infected cells. However P35
replacements of CrmA in CPV infections of LLC-PK1 cells were similar to wt CPV
infections and did not induce apoptosis as analyzed morphologically (Fig. 13) and
biochemically (Fig. 15).


86
However, neither SERP2 nor P35 can control the inflammatory cell influx within
membranes and recombinant CPV virus in which P35 or SERP2 substitute from CrmA
each produce white pocks. We also failed to detect caspase-1 activity within infected
CAMs under any conditions, which suggests that CrmA may function to inhibit
inflammation through a novel target or mechanism. The natural target for CrmA is yet to
be elucidated. The implications to these results are discussed.


190
Models of Inflammatory Response on CAMs
We propose three possibilities to explain the inflammatory response on CAMs.
First is the possibility that our observations are due to species specificity. In the case of
SEPR2 replacements of CrmA, this would not be surprising. We have observed that
during infections of pig kidney cells, SERP2 was unable to inhibit the activation of
caspases (Fig. 20). However, SERP2 was able to inhibit caspase activation during
infections of chicken cells (Figs. 25 and 26). Therefore it is apparent that species
specificity could play a role in the function of SERP2. On the other hand P35 functioned
consistently as an inhibitor of apoptotic induction and caspase activation in both the pig
and chicken systems (Figs. 13, 15, 25 and 26), an observation consistent with reported
properties of P35 functioning in heterologous systems (250). Since neither P35 nor
SERP2 were able to prevent inflammation within the context of CPV infections of
CAMs; there does exist the possibility of species specificity of chicken caspases to P35
and SERP2 (assuming that CAM inflammation is caspase mediated).
Second, there is some evidence in literature for a possible role played by CrmA in
arachidonate metabolism during virus infection. It has been shown that orthopoxviruses
such as CPV require arachidonate metabolites during replication in vivo and in vitro
(303). Blocking the lipoxygenase pathway (but not the cycloxygenase pathway)
prevented pock formation on CAMs and impaired virus replication in tissue culture.
While infection of chicken embryo fibroblasts with CPV up regulated 15-lipoxygenase
and 12-lipoxygenase product formation, deletion of CrmA in CPV caused a dramatic
increase in the formation of products similar to 14(R),15(S) diHETE
(dihydroxyeicosatetraenoic acid), 15HETE and 12HETE (305). Since poxviruses have


186
CPV during infections of CAMs. We did not expect both P35 and SERP2 recombinant
viruses to produce white, inflammatory pocks containing activated heterophils (Figs. 21,
22 and 23). However, P35 and SERP2 proteins function in the context of CPV to restore
virus yields within pocks to wild type levels unlike in the case of the null mutant or the
CrmA D303A mutant viruses (Fig. 24). The restoration of virus yields in the case of both
P35 and SERP2 recombinant virus infections means that inflammation does not
necessarily correlate with reduction in pock titers. P35, SERP2 and CrmA block the
induction of apoptosis on CAMs, whereas pocks from the CrmA null mutant and CrmA
D303A mutant virus infections induced a caspase-3-like activity as seen by the ability to
cleave Ac-DEVD-AMC (Fig. 26). These results correlate with virus yields from pocks
(Fig. 24) confirming our suspicion that reduction in virus yields was a result of failure to
control apoptosis and not due to an inflammatory response. This also opened up the
possibility that the regulation of inflammation and the regulation of apoptosis by CrmA
were two distinct functions that may not be dependent on caspase inhibition alone. Thus
P35 and SERP2 replacements of CrmA in CPV are able to function as inhibitors of
caspase activation but do not inhibit the induction of inflammation within pocks.
Inflammation During CPV Infection may not be due to IL-lp or IL-18
It has been assumed that the lack of inflammation within wt CPV pocks is due to
the ability of CrmA to inhibit the proinflammatory caspase-1 (304) which can cleave
proIL-ip and/or proIL-18 which would lead to formation of the proinflammatory
cytokines IL-1 (3 and IL-18 (16). However, the data presented here as well as some
published data argue against this hypothesis.


197
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57
Apoptosis in Avian Species
Apoptosis in the avian system is less well understood compared to the information
available from the mammalian system. Both mitochondrial mediated and DR mediated
pathways of apoptotic induction have been observed in birds. Most of these studies have
been done in chicken, quail and duck. A number of avian death receptors have been
identified to date. The first avian death receptor protein TVB was identified as the
receptor for avian leucosis sarcoma viruses (49). TVB is 37% identical to human DR-5
death receptor and has been shown to function via its death domain (48, 49). The chicken
B6 (chB6) molecule, widely used as a B lymphocyte marker, does not contain any
apparent DDs, but has been shown to induce apoptosis when cross-linked with antibodies
or overexpressed in murine cells (115, 317). The chicken homolog to human DR-6 is
76% identical to its human counterpart and contains a death domain and two TRAF
binding sites (45). Elevated levels of DR-6 protein correlated with apoptotic induction in
the hen ovary (45). Characterization of partial cDNA sequences of chicken Fas and TNF-
RI have shown 37% and 42% identity to the human homologs respectively (46).
Members of the bcl2 family have also been identified in chickens. The chicken bcl-xl
homolog functions to protect hen granulosa cells from apotosis in the phosphorylated
state (177). The bcl-2 homolog Nr-13 was originally identified as an antiapoptotic
protein activated upon infection by Rous sarcoma virus (124). Purified Nr-13 is able to
bind cytochrome c with high affinity suggesting that it blocks the formation of the
mitochondrial apoptosome involving cytochrome c and Apaf-1 thereby regulating the
induction of apoptosis (265). An IAP protein has also been identified in chickens.


84
In Vivo Role of CrmA During CPV Infections of CAMs
Apoptosis and inflammation are both important mechanisms by which the host
limits and clears viral infections. The results from this project will help gain a better
understanding of viral pathogenesis in terms of both virus-host interactions and the ability
of virus to modulate the host immune system during an infection.
We use the infection of CAMs of embryonated eggs as a model to study the role
of CrmA on the innate immune response including inflammation and apoptosis. The 10
day old CAM is an excellent model to study an acute inflammatory response to infection.
At this stage of embryonic development there is an absence of circulating mature B or T
cells (63,247), minimal complement protein (116) and the inflammatory response due to
CPV infection is mainly mediated by heterophils (analogous to mammalian neutrophils)
and monocytes (112). In order to understand the role played by CrmA during a CPV
infection of the CAM, we addressed the following questions (i) does CrmA prevent white
pock formation by protease inhibition, (ii) if so, is the function of CrmA solely dependent
on inhibition of caspases and (iii) can serpin homologs found in different poxviruses
function similarly?
The first question was addressed by mutating the PI Asp residue in the CrmA
RCL to alanine (D303A), which should abolish serpin function with the predicted
consequence of inducing white pocks. Such a result would also mean that an intact
PI Asp is critical for CrmAs function as an inhibitor of inflammation. The second
question was addressed by replacing CrmA in CPV with other caspase inhibitors such as
P35 of baculovirus or SERP2 from myxoma virus. If the in vivo role of CrmA was to
only inhibit caspases, then P35 and SERP2 should completely replace the function of


64
Caspase Independent Induction of Apoptosis
Apoptosis in the absence of active caspases seems to represent a pathway
conserved in slime mold (Dictyostelium), worms (C.elegans) and mammals (234, 297).
Two proteins have been identified to date that are released from mitochondria and are
postulated to play a role in apoptosis independent of caspase activation. The first is a
novel mitochondrial cell death inducer called the apoptosis inducing factor (AIF) (393).
Mammalian AIF is a 57 kDa flavoprotein that shares homology to oxidoreductases from
vertebrates (Xenopus laevis), non-vertebrates (C.elegans), plants, fungi and bacteria
(234). It was originally discovered as the factor able to induce caspase independent
chromatin condensation and DNA fragmentation in isolated nuclei, but itself has not been
seen to exhibit DNAse activity (393). AIF can also cause permeablization of
mitochondrial membranes and induce release of cytochrome c suggesting a role in a
positive feedback loop with the apoptosome pathway (393). Deletion of AIF in mice
causes embryonic lethality (182). While cells from knockout mice remain sensitive to
apoptosis in response to serum withdrawal, they are resistant to apoptosis induced by the
oxidative stress inducer menadione (182). Since AIF is also an oxidoreductase, it is
unclear whether observations seen in KO mice were due to loss of apoptotic activity
alone or due to the elimination of oxidoreductase activity as well.
The second toxic molecule implicated in caspase independent apoptosis is
endonuclease G (EndoG), which, like AIF, is also released from mitochondria (309).
EndoG is a 30 kDa nuclease, which was originally linked to mitochondrial DNA
replication based on its location and substrate specificity (74). Unlike CAD/DFF, EndoG
is able to induce nucleosomal DNA fragmentation independent of caspase activation


OpNPV
Orgyia pseudotsugata nucleopolyhedrovirus
P35
anti-apoptotic protein from baculovirus
PARP
poly(ADP) ribose polymerase
PBS
phosphate buffered saline
PCD
programmed cell death
PR-3
proteinase-3
PT
permeability transition (mitochondrial)
RPV
rabbitpoxvirus
RCL
reactive center loop
SAPK
stress activated protein kinase
SERP2
serine proteinase inhibitor-2 from myxomavirus
SDS
sodium dodecyl sulfate
SPI-1
serine proteinase inhibitor-1 from poxviruses
SPI-2
serine proteinase inhibitor-2 from poxviruses
SPI-3
serine proteinase inhibitor-3 from poxviruses
SPI-7
serine proteinase inhibitor-7 from swinepoxvirus
STO
mouse fibroblast cell line
TACE
TNF-a converting enzyme
TDS
transient dominant selection
TIR
Toll/IL-IR domain
TLR
Toll like receptor
TNF
tumor necrosis factor
TRAF
TNF receptor associated kinase
Xlll


200
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Cu
107
Figure 24. P35 and SERP2 restore viral yields from infected CAMs. Individual
pocks from infected 11 day old CAMs were isolated at 3 days post infection. Pock
homogenates were plaque assayed on CV-1 cells. Viral plaques were counted 3 days
later. The results are expressed as plaque forming units (PFU)/pock and are averages of
viral titers from 12 pocks. Bar numbers: 1, CPV; 2, CPVAcrmA::lacZ; 3,
CPVAcrmA::P35; 4, CPVAcrmA::SERP2; 5, CPVCrmA D303A. Error bars are standard
deviations of the mean. denotes P < 0.05 by Students t test compared to CPV.


95
DNA Sequencing
Sequencing reactions were performed using 0.5 pg of plasmid DNA or 2pg of
genomic DNA, 4 pmols of primer, 3 pi of ABI Prism Dye Terminator Cycle Sequencing
Ready Reaction kit mix with AmpliTaq DNA polymerase (Perkin Elmer, Foster City,
CA), 2 pi of sequencing core reaction mix (University of Florida ICBR DNA Sequencing
Core Laboratory, Gainesville, FL) and water in a final reaction volume of 20 pi. Twenty-
five thermal cycles carried out according to manufacturers instructions (ABI Prism) in a
PTC-100 Thermal Controller (MJ Research Inc.). Each reaction was centrifuged in a
Millipore Ultrafree MC spin column (Millipore, Bedford, MA) to purify the DNA. The
resulting eluate was dried under vacuum, and the sequence was determined at the
University of Florida ICBR DNA Sequencing Core Laboratory, Gainesville, FL
following gel electrophoresis and analysis using an ABI 373 DNA sequencer.
Sequence Analysis and Database Search
Sequence searches were done using the BLAST search at the NCBI website
http://www.ncbi.nlm.nih.gov/BLAST/. Sequence comparisons and alignments were
performed using the Gap and Pileup programs of University of Wisconsin Genetics
Computing Group (GCG).
Recombinant Virus Construction
Transfection of Infected Cells using Liposomes
CV-1 cells were grown to 80% confluency in 6-well dishes in MEM with Earls
salts with 5% FBS. Medium was removed, and the cells were infected with virus (either


112
Measurement of Reactive Oxygen Intermediates
The CAMs from eggs were harvested and washed twice with PBS. The
membranes were then incubated in 0.1% nitroblue tetrazolium (NBT) (Sigma Chemical
Co.) in PBS at 37C for 1 hour. The NBT solution was removed, membranes were
washed once with PBS and then scanned at 600 dpi. The NBT is reduced to dark blue
insoluble formazan within pocks indicating the presence of reactive oxygen
intermediates.
MTT Reduction Assay
This assay quantitates the amount of reactive oxygen intermediates that may be
present within CAMs. Confluently infected membranes harvested at 48 hours post
infection were excised. Approximately 20mg pieces of membranes were incubated in
200pl of PBS containing 5 mg/ml 3,(4,5-dimethylthiazoI-2yl)-2,5-diphenyltetrazolium
(MTT) for 30 minutes at 37C. The MTT solution was removed, and the reduced
formazan was extracted by grinding the membranes with a microfuge pestle in 200pl of
dimethyl sulfoxide (DMSO) (Sigma). The dissolved formazan was clarified in microfuge
tubes by centrifugation at 12,000 x g for 2 minutes. The absorbance of the resulting
solution was read at 550nm. The absorbance values were expressed as OD/mg of tissue.
Virus Infectivity Assay
Individual virus pocks were isolated and excised from infected membranes. The
pocks were ground using a microfuge pestle in 500pl of PBS, and the cells lysed by three


90
Table 3. List of Primers
Primer
Sequence (5-3)
GM17
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA
GM18
TGCCTAGAATTCCATGGTAATCGATATTGGTCGTGT
GM19
TCG AT C G A ATT C C AT GGC A ATCG ATTTT GTT GT
GM20
ATCGATCGAATTCCCGGGCATATGCCATTTTTTTTAAAAAAAAT
AGAAAAAACATG
GM21
ATCGTACGAATTCCCGGGCATATGATCACATTCTTAATATTAG
AATATTAG
GM22
TGCTACAAGCTTGATGAACACTGATTCCGCATC
FS1
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA
FS74
TACGT C ACCC GGGTT ATTT A ATT GT GTTT A AT ATT AC
FS88
C ACGACC AAT ATCGATT ACT AT GT GT GTAATTTTTCCGG
FS89
C AAC AAAATCG ATT GCC AT GTGT GT AATTTTTCCGG
FS90
AGT AATCGAT ATT GGTCGT G
FS91
GGC AATCG ATTTTGTT G
FS129
GTCAGATCCATGGTTGAATTCCGAGCTTGGCTG
FS130
AGTCAACGCCCGGGTACGCTCACAGAATTCCCG
FS275
TGTGCGCTGGTGGCAGCATGCGCATCAACAGTTACA
FS307
T ACGTCC AT GGAT ATCTTC AGGGAAA
FS308
C AGCTACCCGGGTT AT AATTAGTT GTT GGAG AGC
FS367
ACGTAACC AT GGCT GC AGAAC AAGT AG
FS368
GTCACGGATCCCGGGCTAGTTCTGGTTTTGAAC
FS401
ACGTAGCCATGGCGTTCGTTCCCGACCTG
FS402
GCATTCGGATCCTCAGCGCCCACTTAGCT
FS403
AGT CAT GAATTC AGT AT ACC ACCTGTTAT
FS408
CTGCACGGATCCCAATCACGTTATACTAATAGTAAC
FS409
GCTTACTC AT GAGCTGTG AAG AGA
FS410
ACTTCGGGATCCTGATCATAGGTTGTGCCT
FS413
ACTCCCTGCAGTCATTAATCATTATCCGCTCCTCG
FS414
TCAGACTGCAGCCCGGTAGTTGCGATATAC
FS415
TGACGCCCGGGAGATTAGCGACCGGAGATT
FS416
AGCTGCCCGGGCGTTGAGACCTCCCACAACG


52
The Bcl-2 family consists of more than 12 proteins that have been divided into
three groups based on structural and functional similarities (2, 17). Members of the anti-
apoptotic first group including Bcl-2 and Bc1-xl, contain four conserved Bcl-2 homology
(BH) domains (BH1-BH4). The C-terminal hydrophobic tails localize the proteins to the
outer mitochondrial surface while the major portion of these proteins face the cytoplasm.
The second group consists of pro-apoptotic members including Bax and Bak,
characterized by three BH domains and the C-terminal hydrophobic tail. Members of the
third group are diverse and pro-apoptotic including Bid and Bik, containing only the BH3
domain. Bcl-2 family members can form homodimers, but more importantly the pro- and
anti-apoptotic members interact to form heterodimers. Large numbers of such
heterodimers can be found in the cell. Ultimately the levels of pro- versus anti-apoptotic
family members determine the fate of the cell. An excess of pro-apoptotic Bcl-2 family
members causes cell death conversely the presence of larger numbers of anti-apoptotic
members is protective.
There are a number of models to explain the mechanism of Ai|/m disruption
mediated by the formation of permeability transition pores (PT) that allow the dissipation
of inner membrane ion gradients (147). Since Bcl-2 members oligomerize, they could
potentially form channels to facilitate protein transport. Alternatively Bcl-2 family
members could interact with other proteins to form channels. The voltage dependent
anion channel (VDAC) can bind several Bcl-2 members that regulate its activity (147).
Similarly the adenine nucleotide translocator protein (ANT) has also been implicated to
play a role in Bax induced apoptosis (244). And finally it has been proposed that Bcl-2
members themselves induce the rupture of mitochondrial membranes thereby disrupting


139
A.
1 2 3
Serp2*
34 kDa
Figure 18. Expression of SERP2 and CrmA D303A recombinant proteins. Extracts
were made from LLC-PK1 cells (A) or CV-1 cells (B) infected at an MOI of 10,
harvested at 16 hours post infection, subjected to SDS-PAGE and immunoblotted with
antisera to (A) Serp2 and (B) CrmA. Lanes: 1, CPVAcrmA::SERP2; 2, wt CPV; 3,
Mock; 4, Mock; 5; wt CPV; 6, CPVAcrmA::lacZ; 7, CPVCrmA D303A. Arrows indicate
the position of migrating proteins as follows: Serp2 34 kDa; CrmA -38 kDa and CrmA
D303A 40 kDa. Immunoblots indicate that the recombinant proteins are being
expressed from during virus infection.


170
Z-VAD-FMK may be responsible for proIL-18 processing in CAMs infected with
CPVAcrmA::lacZ.
The results of Figs. 29 and 31 suggest that the chicken proIL-18 processing
activity present in CPVAcrmA::lacZ infections is not due to caspase-1 but rather a
terminal caspase-3-like enzyme present in the apoptotic extracts. In order to eliminate
the possibility that the extracts might contain an endogenous caspase-1 inhibitor, we
added exogenous caspase-1 to CPVAcrmA::lacZ lysates and assayed for chicken proIL-
18 processing activity either in the presence or absence of peptide substrates (Fig. 31,
lanes 7-10). Samples containing exogenously added caspase-1 activity together with the
endogenous caspase-3-like activity present in the CAM extract, fully processed precursor
IL-18 to the mature form (Fig. 31, lane 7). Comparing lanes 7 to 10 in Figure 31, we
concluded that exogenously added caspase-1 activity and any endogenous caspase
activity in the CPVAcrmA::lacZ extracts could be blocked by Z-VAD-FMK (Fig. 31,
lane 10). In the presence of Ac-WEHD-CFIO, which inhibits caspase-1, the remaining
caspase-3-like activity in the extracts remained and produced cleavage (Fig. 31, lane 8).
Conversely Ac-DEVD-CHO blocked caspase-3-like activity in the CPVAcrmA::lacZ
CAM extracts, though the remaining exogenously added caspase-1 activity results in
chicken proIL-18 cleavage (Fig. 31, lane 9). We conclude that the chicken proIL-18
processing activity seen in CPVAcrmA::lacZ extracts is due to a terminal caspase-3-like
activity and not due to caspase-1. Furthermore the white, inflammatory pocks generated
from CPVAcrmA::P35 and CPVAcrmA::SERP2 are not likely the result of caspase-1
mediated proIL-lp or proIL-18 cleavage.


165
Chicken
Pro-IL-18
Processed
Figure 29. P35 and SERP2 function like CrmA to prevent processing of chicken
proIL-18 in CAMs. 3iS labeled chicken proIL-18 proteins made in vitro were incubated
with either buffer (lane 1), 1U of human caspase-1 (lane 2) or with 200 pg of extracts
from confluently infected CAMs harvested 48 hours post infection. Lanes: 1, Buffer; 2,
1U caspase-1; 3, CPV; 4, CPVAcrmA::lacZ; 5, CPVAcrmA::P35; 6,
CPVAcrmA::SERP2; 7, CPVCrmA D303A. The protein mixtures were resolved by
electrophoresis on 10% SDS-PAGE and visualized by autoradiography. Radiolabeled
peptides are: chicken proIL-18 23 kDa; processed form 19 kDa.


8
The initiation of signaling by TLRs could occur directly following receptor
engagement as in the case of TLR4 (254) (Figure 1). Alternatively the ligand may need
to be processed prior to receptor interaction as seen with Drosophila Spatzle, which
requires cleavage by serine protease Easter in order to activate Toll (221). The signaling
events following TLR engagement seem to be conserved from flies to mammals.
Signaling by TLR is depicted in Figure 1. Similar to IL-1 binding IL-1R, MyD88, an
adapter molecule that contains a TIR domain, is recruited to the receptor complex where
it causes autophosphorylation of serine threonine kinase IRAK (255). Complex protein
kinase interactions result in the activation and translocation of NF-kB to the nucleus
where it induces expression of inflammatory cytokine genes. TLR2, 5, 6 and 9 activate
NF-kB, while TLR2, 4, 6 and 9 can induce the transcription factor activator protein-1
(AP-1) activity (433). TLR2 has been shown to mediate NF-kB activity via PI3 kinase,
suggesting the involvement of GTP binding proteins (18). TLR2 involvement in
apoptosis has also been demonstrated (13, 14). The fact that mutant caspase-8 could
inhibit TLR2 activity suggests the possibility of direct activation of caspases by TLR2.
The inflammatory cytokines TNF-a, IL-1 P, IL-18 and IL-8 that are upregulated by TLRs
(254) will be discussed in detail later. The identification of more signaling events
associated with TLRs will bring to light their different roles in the immune response. The
TLR system is similar to mammalian IL-1R system (293).
Interleukin-1 (IL-1)
IL-1 is one of the earliest cytokines produced in response to cellular damage or
infection. IL-1 p is the prototypical proinflammatory cytokine that belongs to the IL-1


65
(224). It has also been implicated in DNA fragmentation seen in DFF deficient mouse
embryonic fibroblasts following induction of apoptosis by UV-radiation and TNF
treatment (469). A homolog to EndoG in C.elegans has been identified as CPS-6, which
shows similarity in sequence, localization and biochemical properties to mammalian
EndoG (309). Mutant C.elegans deficient in CPS-6 can be rescued by mouse EndoG
(309). The involvement of EndoG in cell death indicates the presence of an apoptotic
pathway, independent of caspase activation that is conserved from worms to mammals.
Although the caspase independent pathway is not completely understood, the
identification of AIF and EndoG clearly supports the existence of an apoptotic program
parallel to caspase activation.
Poxviruses
The Poxviridae are a family of large double stranded DNA viruses that replicate in the
cytoplasm of infected cells. The taxonomy of poxviruses is based on hosts they infect and
include the two subfamilies Chordopoxvirinae containing vertebrate viruses and
Entomopoxvirinae containing the insect poxviruses (Table 2) (271). The
Chorodopoxvirinae comprises eight genera: Orthopoxvirus, Parapoxvirus, Avipoxvirus,
Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. While
there are three genera within Entomopoxvirinae, these viruses may need to be reclassified
based on recent sequence information that suggests that they may not be as closely
related as previously thought (3, 27). Both variola virus (Orthopoxvirus genus) and
molluscum contagiosum virus {Molluscipoxvirus genus) are the only poxviruses to
exclusively infect humans (271).


120
A.
SPI-2
178,373 179,407
RPV Genome
197,732 bp
GM 17
SPI-2 ORF 20
B
GM 18
GM 22
B.
Xbal
LacZ
Hindlll
Xbal ,c Hindlll
SPI-2 Uft Hank (1005)
Figure 8. Construction of recombinant RPV. (A) The location of SPI-2 within the
RPV chromosome is indicated. Left and Right flanks (hatched boxes) of the SPI-2
coding region were amplified using the primers indicated. (B) Illustration of the shuttle
vector used to replace SPI-2 in RPV with either lacZ, or P35 genes. The plasmid pBS-
RgS/A contains the E.coli gpt gene outside the SPI-2 flanks (hatched boxes). The various
genes were cloned into pBS-RgS/A, which was then used to generate recombinant RPVs
by transient dominant selection (97). Restriction sites used for cloning are indicated. See
Materials and Methods (Recombinant Virus Construction) for each individual virus
construct.


83
Alignment of SPI-2 and CrmA
1 mdifreiassmkgenvfispasissvltilyygangstaeqlskyvekee 50
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
1 mdifreiassmkgenvfisppsissvltilyygangstaeqlskyvekea 50
51 nmdkvsaqnisfksinkvygrysavfkdsflrkigdkfqtvdftdcrtid 100
-I 11111-111111111111111111111 11111111111:1
51 dknk...ddisfksmnkvygrysavfkdsflrkigdnfqtvdftdcrtvd 97
101 ainkcvdiftegkinplldeqlspdtcllaisavyfkakwltpfekefts 150
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
98 ainkcvdiftegkinplldeplspdtcllaisavyfkakwlmpfekefts 147
151 dypfyvsptemvdvsmmsmygkafnhasvkesfgnfsiielpyvgdtsmm 200
111111111111111111111.111111111111111111111111111.
148 dypfyvsptemvdvsmmsmygeafnhasvkesfgnfsiielpyvgdtsmv 197
201 vilpdkidglesieqnltdtnfkkwcnsleatfidvhipkfkvtgsynlv 250
I I I I I I I I I I I I I I I I I I I I I I I I I I :: I I I I I I I I I I I I I I I I I I I
198 vilpdnidglesieqnltdtnfkkwcdsmdamfidvhipkfkvtgsynlv 247
251 dtlvksgltevfgstgdysnmcnldvsvdamihktyidvneeyteaaaat 300
I III I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
248 dalvklgltevfgstgdysnmcnsdvsvdamihktyidvneeyteaaaat 297
301 svlvadcastvtnefcadhpfiyvirhvdgkilfvgrycspttnc* 346
II I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I
298 calvadcastvtnefcadhpfiyvirhvdgkilfvgrycspttn*. 342

PI
Figure 7. Comparison of SPI-2 and CrmA peptide sequences. PI position is indicated
by arrowhead. Sequence alignment was performed using GCG Gap program.


227
346. Roy, N., Q. L. Deveraux, R. Takahashi, G. S. Salvesen, and J. C. Reed. 1997.
The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases.
EMBO J. 16:6914-6925.
347. Ruchaud, S., N. Korfali, P. Villa, T. J. Kottke, C. Dingwall, S. H. Kaufmann,
and W. C. Earnshaw. 2002. Caspase-6 gene disruption reveals a requirement for
lamin A cleavage in apoptotic chromatin condensation. EMBO J. 21:1967-1977.
348. Ruffatti, A., R. A. Sinico, A. Radice, E. Ossi, F. Cozzi, M. Tonello, P.
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352. Saraiva, M. and A. Alcami. 2001. CrmE, a novel soluble tumor necrosis factor
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151
Figure 23. Inflammatory pocks show similar levels of oxidative burst. Confluently
infected CAMs were harvested at 48 hours post infection. Reactive oxygen intermediates
were measured by incubating pooled pocks in the presence of MTT for 30 minutes. The
reduced formazan was dissolved in DMSO and absorbance read at 550nm. The results
are expressed as OD 550/mg of tissue. Endogenous NADPH oxidase activity from
uninfected CAMs have been subtracted. Bar numbers: 1, Mock; 2, CPV; 3,
CPVAcrmA::lacZ; 4, CPVAcrmA::P35; 5, CPVAcrmA::SERP2; 6, CPVCrmA D303A.
The results are standard error of the mean of four such experiments. denotes P < 0.05
by Students t test compared to CPV.


147
-NBT
+NBT
Figure 21. Continued


149
Figure 22. Histological examination of pocks from infected CAMs. CAMs infected
with recombinant viruses were harvested 3 days post infection. Individual pocks were
sectioned and stained with hematoxylin and eosin. (A) Mock infected CAMs consist of a
thin ectoderm, approximately 2 cells in thickness, a mesoderm with low numbers of cells
in the extracellular matrix and a thin endoderm, 1-2 cells thick. (B) Mock infected
CAMs featuring individual mesodermal cells at 500 x magnification. (C) CPV infected
pocks show marked ectodermal and mesodermal hyperplasia. Hemorrhage can be seen
between ectodermal and mesodermal layers of the CAM. (D) CPV infected pocks
showning the ectodermal and mesodermal junction at 500 x magnification. (E & F)
CPVAcrmA: :lacZ, (G & H) CPVAcrmA::P35, (I & J) CPVAcrmA::SERP2 and (K & L)
CPVCrmA D303A pocks show ectodermal hyperplasia and extensive heterophilic
inflammation in the mesoderm without evidence of hemorrhage. H-heterophils
(indicated by arrows); E-Ectoderm; M-mesoderm.


11
Figure 1. Signaling by TNF, IL-1R and TLR. (A) TNF binds to TNFRI and causes the
association of TRADD, which recruits RIP and TRAF2 to the receptor complex.
Activation of IKK occurs through the activation of IKK kinases or by RIP mediated
oligomerization of IKK complexes. TNFRI induces cell death when TRADD recruits
FADD and activates caspase-8. (B) IL-1 and TLR receptor complexes which include
MyD88 and IRAKs induce the ubiquitin dependent association of TRAF6. As a result
IKK kinase Takl is activated which phosphorylates and activates the IKK complex.
Abbreviations: TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, TNF receptor
associated death domain; FADD, Fas ligand associated death domain; IKK, I kappa B
kinase; NF-kB, nuclear factor kappa B; IL, interleukin; LPS, lipopolysaccharide; IKK, I
kappa B kinase; IRAK, IL-1 receptor associated kinase, Mai, MyD88 adapter-like,
TRAF, TNF receptor associated factor. From reference 42.


30
macrophages, it was not blocked by antibodies to human TNF-a suggesting structural
differences and/or the presence of other factors in the preparation. Chicken TNF-RI has
recently been cloned and was found to be 47% identical to its human counterpart in the
cytoplasmic death domain (46). In addition a number of death receptors (DRs) related to
TNF-RI have been identified in chickens that are yet to be fully characterized (46).
IL-1
Chicken IL-1 activity was first seen in LPS stimulated spleenocyte supernatants
(140). Chicken macrophages treated with LPS also induced secretion of IL-1 activity
(41). The induction of IL-1 was seen to be dependent on protein kinase C and a
calmodulin dependent kinase. Other studies have also shown that IL-1 secretion by
macrophages is dependent on intracellular calcium levels (65). IL-1 also lowered growth
rates in chicken similar to the effects seen with inflammatory agent Sephadex (195). The
chicken IL-1 P gene was cloned rather recently and a peptide containing residues 106 to
267, the putative mature IL-p sequence, was shown to be biologically active both in vitro
and in vivo (446).
Unlike the mammalian system where maturation of IL-1 P requires caspase-1 to
process the inactive precursor initially at Asp27 (site 1) followed by cleavage at Aspl 16
(site 2), the chicken proIL-1 p sequence does not contain either of the conserved Asp
residues required for cleavage that would release the mature cytokine (395, 446). The
mechanism of IL-1 P maturation in chickens is currently unknown. It remains a
possibility that in chickens, IL-ip does not require processing and maybe active as the
precursor itself. The chicken proIL-ip sequence is similar to proIL-ip sequences from


105
Construction of CPVCrmA D303A
The D303A mutant CrmA coding region was PCR amplified using FS 307 (5-
TACGTCCATGGATATCTTCAGGGAAA-3) containing an Ncol site (underlined) and
FS 308 (5-CAGCTACCCGGGTTATAATTAGTTGTTGGAGAGC-3) containing a
Smal site (underlined). The PCR product was cloned into the Ncol and Smal sites of
pBS-CgS/A resulting in pBS-D303ACgS/A. Using CPVAcrmA::lacZ and pBS-
D303ACgS/A, CPVCrmA D303A was made by transient dominant selection and X-Gal
staining of viral plaques. The viral construct was verified by sequence analysis and for
expression of mutant CrmA by immunoblot.
Construction of CPVAIL-1 pR Virus
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sequence) and corresponds to base pairs 194007-194987 in the CPV genome. The design
of the knockout plasmid was such that the P7 5-gpt cassette that confers resistance to
mycophenolic acid (96) was cloned between the first 260 base pairs of CPV IL-1 p
receptor (left flanking sequence) and the last 223 base pairs of the viral receptor (right
flanking sequence). The left flanking sequence was PCR amplified with Vent
polymerase (Amersham) from CPV genomic DNA using primers FS 403 (5-
AGTC AT GAATTC AGT AT ACC ACCT GTT AT-3 ) containing EcoRI site (underlined)
and FS 413 (5-ACTCCCTGCAGTCATTAATCATTATCCGCTCCTCG-3) containing
PstI site (underlined). The left flanking PCR product was cloned into the EcoRI and PstI
sites of pBluescript II KS(+) (Stratagene) resulting in pBS-CPVIL-lR-LF.


236
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456. Xiang, Y., R. C. Condit, S. Vijaysri, B. Jacobs, B. R. Williams, and R. H.
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essential component of the IL-1 receptor. J. Immunol. 161:5614-5620.


33
Viral Inhibitors of Inflammation
The outcome of viral infections depends on both host and viral factors. The early
resolution of infection is the goal of the mounted innate immune reaction. Not
surprisingly, viruses have been shown to encode a number of molecules that interfere
with the early host innate response. Viral products that specifically function to abrogate
pro inflammatory cytokines and their effects will be discussed here. Cytokine synthesis is
a typical early host response to viral infection. Different viruses target the same cytokine
pathways underlining their importance as potent anti-viral agents and these include TNF-
a, IL-lp, IL-18, the interferons, complement as well as a number of chemokines. The
strategies employed by viruses function to disrupt cytokine transcription/up regulation,
interfere with receptor binding and modulate cytokine signaling.
TNF Inhibitors
Encoding decoy receptors that bind TNF is a mechanism employed by a number
of poxviruses and herpesviruses. Shope fibroma virus encodes the T2 protein that has
been shown to be a TNF receptor homolog that binds both TNF-a and p (249). A
homolog to the T2 protein was also found in Myxoma virus (427). Deletion of Myxoma
virus T2 had no effect on growth of the virus in tissue culture but significantly attenuated
viral lethality in infected rabbits. Cowpoxvirus encodes five different TNF receptor
family homologs CrmB, CrmC, CrmD, CrmE and a soluble CD30 homologue. The
CrmB protein shares homology to TNFRII in the ligand binding region and can bind both
TNF-a and P (162). CrmC also shares homology to TNFRII but only binds TNF-a
(375). CrmD found only in some strains of cowpoxvirus and ectromelia virus was able to


129
hr p.i.
Figure 15. Biochemical changes in LLC-PK1 cells infected by CPV derivatives. Pig
kidney cells were infected at MOI 10 and harvested at various times post infection. Cell
extracts were made and incubated with terminal caspase substrate Ac-DEVD-AMC, a
fluorogenic peptide. Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Ac-
DEVD-AMC cleavage rates were determined at each harvested time point and are
expressed arbitrarily as fluorescence signal units (FSU) per second. Legends
corresponding to the different viruses tested are indicated.


40
Apoptosis
Apoptosis is a crucial step necessary for the clearance of infected cells and
activated neutrophils, to protect areas adjacent to the inflammatory tissue from injury.
Apoptosis or programmed cell death is a distinct mechanism by which cells die and are
dismantled in an orderly fashion (191). This process is a distinct morphological and
biochemical function that involves cell shrinkage, membrane blebbing, degradation of
proteins, chromatin condensation, DNA fragmentation followed by disintegration of the
cell into multiple membrane enclosed vesicles termed apoptotic bodies (454). The
disassembled cell is finally phagocytosis by its neighbors. This form of cell death differs
from necrosis where cells typically lyse by loss of membrane (191). Apoptosis is part of
normal homeostatic mechanism whereby cells die during embryonic development, tissue
turnover and as a means of defense against pathogens. Metazoan development is
characterized by the over production of cells followed by apoptotic induction to cull the
excess cells at later stages of development such that the relative number of cells required
to achieve proper function is maintained. This phenomenon is first characterized in the
nematode Caenorhabditis elegans by Horvitz and others (468). Processes of assembly
and disassembly constantly maintain the normal human bodys approximately 1014 cells.
It is therefore not surprising that inappropriate cell death (either too much or too little)
can give rise to disease conditions. Excessive apoptosis has been implicated in
neurodegenerative disease such as Alzheimers (242, 387) and in tissue destruction that
occurs after vascular occlusions in the brain and heart (34, 62, 286). Similarly the lack of
apoptosis contributes to the development of tumors (2) and autoimmunity such as
autoimmune lymphoproliferative syndrome in humans (107, 339, 439).


12
1 Ra does not bind the third Ig domain on the receptor and cannot induce conformational
change in the receptor that would allow IL-lRAcP docking (357). IL-1 mediated signal
transduction was absent in both IL-1 RE7' and IL-lRAcP'7' mice, suggesting that both the
receptor and accessory proteins were essential components for signaling (77, 126, 212).
The IL-1RII also has three Ig domains but lacks the TIR domain (252). IL-1RII binds all
three forms of IL-1 (252). The lack of the transducing cytoplasmic TIR domain indicates
that it functions as a negative regulator of IL-1 activity. Consistent with this role, IL-
1RII has very low affinity for IL-1 Ra (398). IL-1RII is expressed highly on lymphoid
and myeloid cells where it can be shed and act as a soluble decoy receptor (73). It was
presumed that proteolytic cleavage of the extracellular domains of membrane receptors
results in the soluble forms of these receptors (83). Indeed TNF-a converting enzyme
(TACE) has been shown to shed IL-1 RII from cell membranes (334). Soluble forms of
the type I receptor have also been found in circulation. Patients with rheumatoid arthritis
and sepsis have elevated levels of soluble IL-IRs indicating that these receptors function
to regulate IL-1 activity in vivo (19,125). In areas of local inflammation, tissue necrosis
can release precursor IL-1 p into the extracellular environment where it potentially can be
processed by proteases other than caspase-1. The maturation of IL-1 p mediated by
enzymes other than caspase-1 is a consideration that may be important in the results
presented in this dissertation. Both IL-1RI and II can bind precursor IL-1P and prevent
its processing, indicating the importance of soluble receptors in inflammatory fluids
(398).
The fact that biologically active IL-1P was seen in caspase-1 '/ mice suggested the
existence of other proteases that could process proIL-1P (100). This observation may be


176
Using CPVAIL-ipR, 11 day old CAMs were infected and the lesions were examined 72
hours post infection (Fig. 34). The pocks caused by CPV AIL-1 PR were indistinguishable
from wt CPV pocks (compare Fig.21 and 34) which are hemorrhagic and non
inflammatory as they do not reduce NBT.
Taken together these results further add weight to the hypothesis that the
inflammation seen on CAMs in the absence of CrmA during CPV infections (Fig.21) is
not due to actions mediated by IL-1 p. Results from our study using recombinant CPV
infections are summarized in Table 7.


7
found to control anti-fungal response in flies (221). The intracellular domain of Toll was
found to be homologous to a similar region in human interleukin-1 receptor (IL-1R)
(293). Homologs to IL-1R have since been discovered, that now belong to the Toll-like
receptor (TLR) superfamily.
TLRs are transmembrane proteins that contain leucine rich repeat (LRR)
extracellular domains and Toll/IL-IR (TIR) cytoplasmic domains. The mammalian IL-1
system will be discussed in further detail later. Similarities exist between the fly Toll and
mammalian IL-1 system. Engagement of both receptors by the respective ligands results
in the activation of nuclear transcription factors. In addition to Spatzle, there also exist
adapter proteins such as Tube and a protein kinase Pelle. To date there are at least nine
Drosophila and ten mammalian TLRs known (172). In addition to the Toll ligand,
Spatzle, there are six other Spatzle-like proteins (281). TLR4, the first mammalian TLR
identified was shown to activate NF-kB and induce IL-1, IL-6 and IL-8 genes (254).
Studies with the mouse homolog of TLR4 demonstrated its role in the recognition of LPS
and gram-negative bacteria (172). TLR2 and TLR5 also recognize bacterial components
such as peptidoglycan, LPS, lipids from trypanosomes and flagellin (150, 362), (53),
(141). TLR2 binding has been shown to cause internalization of the receptor bound
ligand to the macrophage phagosome (141,426). Whether this is an exception or a rule
for most TLRs is to be seen. Experimental evidence also suggests that TLR1, 2 and 6
may function in a cooperative manner (138, 301). This suggests that heterodimerization
of receptors could in theory increase the repertoire of ligand specificities. TLR9 is
thought to reside intracellularly on lysosomes where it recognizes unmethylated CpG
motifs in bacterial DNA and induces ThI response (145).


77
of the SERP2 gene in myxoma virus leads to attenuation of disease as evidenced by
survival of infected rabbits (257). SERP2 has been reported to inhibit the influx of
inflammatory cells into the site of primary inoculation as well as prevent apoptosis of
infected lymphocytes in the rabbit model. While recombinant cowpox virus expressing
SERP2 instead of CrmA failed to block the induction of apoptosis in pig kidney cells,
SERP2 in the context of myxoma virus does inhibit apoptotic induction during infection
of rabbit kidney cells (424). The importance of the PI residue in SERP2 is evidenced by
the observation that mutant viruses, in which the PI Asp is changed to Ala, cause an
attenuated disease (Amy MacNeill and Richard Moyer unpublished). If SERP2 were to
function simply as a caspase inhibitor in vivo, then a recombinant myxoma virus
expressing CrmA instead of SERP2 should produce similar effects as the wt myxoma
infection. On the contrary CrmA replacements of SERP2 in myxoma virus causes only
-60% mortality with a marked difference in primary lesion pathology (Amy MacNeill
and Richard Moyer unpublished). Thus SERP2 is an important virulence factor for
myxoma and deducing its natural target would provide clues to its role during myxoma
infection as well as its function as a serpin.
Cytokine Response Modifier A (CrmA)
CrmA was the first viral serpin molecule identified (316). It is an example of a
cross class inhibitor in that it has been shown to inhibit both the serine protease granzyme
B as well as members of the caspase family of cysteine proteases (201). CrmA was first
identified based on its ability to alter lesion morphology on chicken chorioallantoic
membranes (CAM). CPV produces characteristic red, hemorrhagic, non-inflammatory


166
recognized by human caspase-1. No cleavage was observed when proIL-18 was
incubated with extracts from CPV infected CAMs (Fig. 29, lane 3). However,
CPVAcrmA::lacZ and CPVCrmA D303A infected CAM extracts both contain an activity
able to cleave chicken proIL-18 (Fig. 29, lanes 4 and 7), whereas extracts from CPV,
CPVAcrmA::P35 or CPVAcrmA::SERP2 infections failed to process chicken proIL-18
(Fig. 29, lanes 3, 5 and 6). We conclude that P35 and SERP2 function like CrmA in
preventing chicken proIL-18 processing activity even though the respective recombinant
viruses still produce inflammatory pocks. Therefore IL-18, like IL-ip is unlikely to be
processed in this system. Hence, neither cytokine is likely to be the mediator of
inflammation on CAMs caused by either CPVAcrmA::P35 or CPVAcrmA::SERP2
infections.
Chicken ProIL-18 Processing Activity in CPVAcrmA Lysates is Blocked by Caspase
Specific Peptide Inhibitors.
Although collectively among the viruses tested there is no correlation with control
of inflammation and proIL-1 p or proIL-18 cleavage, extracts from CPVAcrmA:: lacZ and
CPVCrmA D303A virus infected CAMs do contain an activity which can process proIL-
18. We sought to provide further evidence that the processing activity is not related to
caspase-1. Therefore, we first assayed the ability of chicken proIL-18 to be cleaved by
purified caspases-1 or -3 and also verified the specificity of caspase inhibitors towards
caspase-1 (Fig. 30). As expected, chicken proIL-18 was readily cleaved by caspase-1.
Furthermore, that reaction was blocked by the caspase-1 inhibitor Ac-WEHD-CHO and
the general caspase inhibitor Z-VAD-FMK but unaffected by the caspase-3 inhibitor Ac-


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196


102
Construction of Recombinant CPV Expressing SPI-2
The upstream flanking sequence of the CrmA coding region was PCR amplified
with Vent polymerase (Amersham) from CPV genomic DNA using primers GM 17 (5-
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-31 containing
Xbal and Notl sites (underlined) and GM 19 (5-
TCGATCGAATTCCATGGCAATCGATTTTGTTGT -3) containing Ncol and EcoRI
sites (underlined). The PCR product was cloned into the Xbal and EcoRI sites of
pBluescript II KS (+) (Stratagene) resulting in pBS-CLF. The downstream flanking
sequence of the CrmA coding region was PCR amplified with from CPV genomic DNA
using primers GM 21 (5- ATCGTAC
GAATTCCCGGGCATATGATCACATTCTTAATATTAGAA TATTAG-3) containing
EcoRI, and Smal sites (underlined) and GM 22(5-
TGCTACAAGCTIGATGAACACTGATTCCGCATC-3) containing a Hindlll site
(underlined). The right flank PCR product was cloned into the EcoRI and Hindlll sites of
pBS-CLF resulting in pBS-CCF.
The P7.5gpt cassette, which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the EcoRI site of pBS-CCF resulting in
the shuttle vector pBS-Cgpt. By transient dominant selection, CPVAcrmA::Eco.gpt was
created using pBS-Cgpt and wt CPV.
The SPI-2 gene from pTMlHisSPI-2 (Peter Turner unpublished) was re-cloned
into the Ncol and Smal sites of pBS-CCF to generate pCSG. The recombinant
CPVAcrmA:SPI-2 virus was created by reverse selection on STO cells using 6-
thioguanine (170) using pCSG and CPVAcrmA::Eco.gpt.


114
Construction of Plasmids Containing Chicken ProIL-ip or ProIL-18
Since caspase-1 processes proIL-ip and proIL-18 to the mature cytokines, we
wanted to test CAM extracts for the ability to process radiolabeled chicken cytokine
precursors transcribed and translated in vitro. Plasmid cDNA clones containing the
coding sequence of chicken proIL-1 p or proIL-18 were kindly provided by Peter Staeheli
(University of Freidburg, Germany). The chicken proIL-ip gene was PCR amplified
from the original plasmid cDNA clone using Vent polymerase (New England Biolabs)
with primers FS 401 (5-ACGTAGCCATGGCGTTCGTTCCCGACCTG-3) at the 5
end of the open reading frame containing an added Ncol site (underlined) and FS 402 (5-
GCATTCGGATCCTCAGCGCCCACTTAGCT-3) at the 3 engineered to contain a
BamHI site. Similarly the chicken IL-18 gene was amplified with primers FS 409 (5-
GCTTACTCATGAGCTGTGAAGAGA-3) at the 5 end of the open reading frame
containing an added BspHI site (underlined) and FS 410 (5-
ACTTCGGGATCCTGATCATAGGTTGTGCCT-3) at the 3 engineered to contain a
BamHI site. The PCR products were cloned into the Ncol and BamHI sites of pTMl
(272) resulting in pTMlchIL-ip containing the chicken proIL-ip coding sequence and
pTMlchIL-18 containing the chicken proIL-18 sequence. The plasmid constructs were
oriented such that the open reading frames could be expressed from the T7 promoter of
with each plasmid. 35S-labelled chicken proIL-ip and proIL-18 were synthesized using
the T7 Quick Coupled Transcription/Translation System (TNT) (Promega Corporation)
and Trans 35S-Label (ICN) as the source of [35S] methionine according to the
manufacturers instructions.


152
P35 and SERP2 Restore Viral Yields from Infected CAMs
When compared to pocks produced by wt CPV, the pocks produced by
CPVAcrmA::lacZ are inflammatory and have been shown to produce far less virus (306).
It was presumed that the reduction in virus yields was due to the induction of an
inflammatory response in the CAMs. If this were true we would predict that all the
inflammatory pocks produced by the various recombinant CPV should also have lowered
virus yields. We have measured the ability of P35 and SERP2 to restore virus yields.
Following infection, individual pocks were harvested, the pocks homogenized and the
virus titers determined on CV-1 cells. The resulting virus yields were expressed as plaque
forming units (PFU)/pock (Fig. 24). Wild type CPV pocks yield approximately 5 x 106
PFU/pock, while CPVAcrmA::lacZ yields were at least a ten fold lower (4 x 105
PFU/pock) consistent with previous reports (306). Yields of virus from CPVCrmA
D303A pocks were similar to those of CPVAcrmA::lacZ, a result again consistent with
the prediction that the PI Asp of the RCL is required for CrmA to function as a protease
inhibitor during CPV infection in vivo. Both P35 and SERP2 recombinant viruses are
able to restore virus yields to wild type levels. Hence the two proteins P35 and SERP2
can partially substitute for CrmA function within the context of a CPV infection. These
results show that the reduction in pock titers and the white, inflammatory pock phenotype
are not linked and reflect two different functions of CrmA. The restoration of virus
yields, despite the presence of inflammation, suggests that CrmA, might function within
the CAM to control apoptosis and that induction of apoptosis, rather than inflammation in
the absence of CrmA leads to lowered virus yields. It is important to note that in cell


92
following isolation of plasmid DNA from cells using QIAfilter Maxi-prep kits (Qiagen,
Valencia, CA).
Viral DNA Preparation
Confluent monolayers of CV-1 cells were infected with either RJPV, CPV or their
derivatives, as described in viral stock preparation. After complete CPE, the supernatants
were collected following centrifugation of cells at 1000 x g for 4 minutes. The
supernatants were transferred to 30ml polypropylene tubes, and extracellular enveloped
virus (EEV) was pelleted by. The viral pellet was re-suspended in water (100 pi per 150
mm dish) containing 1% SDS (Sigma Chemical, St.Louis, MO) and 1 mg/ml proteinase
K (Sigma) and incubated overnight at 50C.
The solutions were mixed with an equal volume of phenol/chloroform and
centrifuged at 16,000 x g for 4 minutes. The aqueous layer was mixed with chloroform
and centrifuged at 16,000 x g for 4 minutes. DNA in the aqueous phase was precipitated
with 2 volumes of 95% ethanol and 1/10 volume of 3M sodium acetate (ph 5.3) at -20C
overnight. DNA was centrifuged at 16,000 x g for 4 minutes in microfuge tubes, and the
pellets were washed once with 70% ethanol and re-suspended in water (100 pi per
150mm dish). The DNA was quantified using a fluorimeter.
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Extracts of infected cells were prepared by resuspending cell pellets in Triton
lysis buffer (100 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% v/v Triton X-100) and lysed
by 3 freeze-thaw cycles. Cell debris was pelleted at 1500 x g for 10 minutes and the


99
Reverse Selection of Recombinant Virus using 6-Thioguanine
Expression of Eco.gpt can be used to generate recombinant virus in the presence
of 6-thioguanine (6TG), where selection is performed for virus lacking the Eco.gpt gene
(170). 6TG is a purine analog and is toxic to cells or virus expressing guanine
phosphoribosyl tranferase. Therefore cells such as STO cells used for reverse selection
lack the GPT gene. Briefly, STO cells were grown in the presence of 6TG in 6-well
dishes (0.2 mM for RPV and 0.4 mM for CPV). Following liposome-mediated
transformation of infected CV-1 cells as described earlier, the virus mixture was plated
on STO cells in the presence of 6TG. Viral plaques were stained 72 hours post infection
with neutral red. Typically four virus plaques were selected during the first viral pick and
purified five times by plaque purification in the presence of 6TG on STO cells. At this
stage, each independently derived virus clone was grown on CV-1 cells in 60 mm dishes,
and viral DNA was prepared. Identification of recombinant virus was performed
following PCR amplification of the foreign gene and confirming the lack of Eco.gpt
gene. A list of plasmids and viruses constructed in this study is listed in Tables 5 & 6.
Construction of Recombinant RPV Expressing CrmA
The upstream flanking sequence of the SPI-2 coding region was PCR amplified
with Vent polymerase (Amersham) from RPV genomic DNA using primers GM 17 (5-
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-31 containing
Xbal and Notl sites (underlined) and GM 18 (5-
TGCCTAGAATTCCATGGTAATCGATATTGGTCGTGT -3) containing Ncol and
EcoRI sites (underlined). The PCR product was cloned into the Xbal and EcoRI sites of


189
proIL-18 processing activity observed in CPVAcrmA::lacZ extracts was due to a caspase-
3like activity and not due to caspase-1.
IL-1 p is produced by a number of cell types during tissue injury (85) and is
controlled in two ways during orthopoxvirus infection. In addition to regulating IL-1 P at
the post-translational level by blocking caspase-1 using CrmA, orthopoxviruses also
encode a soluble IL-1 P receptor (vIL-1 PR). It has been shown in the intranasal mouse
model of infection that vIL-1 PR rather than SPI-2 (CrmA homologue in vaccinia virus)
controls the febrile response mediated by IL-1 P (192). In order to verify this observation,
we deleted the vIL-1 PR in CPV and the resulting virus was used to infect CAMs (Fig.
34). If IL-1 P were to be involved in the inflammation on the CAMs, one would expect
CPV AIL-1 pR infections to produce white, inflammatory pocks. We constructed an
insertional mutant CPVAIL-ipR and used this virus to infect 11 day old CAMs.
CPV AIL-1 PR virus produced red, hemorrhagic, non-inflammatory pocks like wild type
CPV, which did not reduce NBT (Fig. 34). This observation strengthened our hypothesis
that the inflammation on CAMs was not mediated by IL-1 p. Since the inflammatory
response on CAMs is mediated by heterophils during CPVAcrmA::lacZ infection, it is
probable that heterophil chemoattractants other than IL-1 P are involved. The potential
candidates would include leukotrienes, TNFa, IL-8, IL-6 and to a lesser extent IL-12
since this requires a functional cell mediated response, which is absent in 10 day old
chick embryos.


48
functionality of human caspase-12 has been lost since it is reported to contain a number
of mutations that prevent it from being expressed as a full length protein (105).
Apoptotic Initiator Caspases-2, -8, -9, and -10
The first caspase related disease condition described in humans is autoimmune
lymphoproliferative syndrome (ALPS) type II, an autoimmune disorder linked to
mutations in the initiator caspase-10 (439). Initiator caspases represent members
involved in both pathways leading to caspase activation namely the receptor mediated
extrinsic pathway involving caspase-8 and the intrinsic mitochondrial pathway involving
caspase-9. DR induced apoptosis involves receptor ligand interactions that aggregate and
act as scaffolds for caspase activation often termed the death inducing signaling complex
(DISC) (90). The recruitment of initiator caspases to the DISC involves the DEDs in the
prodomains of caspase-8 and -10 that are involved in binding DEDs on adaptor
molecules such as FADD in the DISC (438). Similarly the CARD found in caspase-2
and -9 prodomains has been shown to associate with CARDs on adaptor molecules such
as RAIDD facilitating the recruitment of caspase-2 to the DISC (5, 88). The induced
proximity model proposes that presence of high local concentrations of procaspase-8 in
the DISC is sufficient to stimulate the autoactivation of caspase-8 (283). This
phenomenon of autoactivation has been noted for caspase-9 and -10 (382, 438). An
alternative hypothesis is that conformational changes induced by binding adaptor
molecules can cause activation of proinitiator caspases (147). Activated caspase-8 can
either cleave the terminal effector caspases-3 and -7 directly or can cleave the


3
cause the infected cell to undergo apoptosis or cell death thus preventing viral replication
and release of progeny. Intracellular pathogens taken up by macrophages can evade lysis
by residing in vesicles that have not fused with the lysosome. A subset to T cells termed
T-Helper 1 (ThI) cells; promote activation of macrophages which allows fusion of their
lysosomes with vesicles thus destroying the invading pathogen. Protection against
extracellular pathogens mediated by antibodies also involves the function of a third
subset of T-lymphocytes termed T-helper 2 cells (Th2). Th2 cells stimulate B-cells to
proliferate and produce antibody to particular antigens. In most cases the adaptive
response confers life long immunity against re-infection by the same pathogen.
Innate Immunity and Inflammation
Inflammation is the reaction of living tissue to any form of injury. Traditionally
inflammation is defined by the Latin words calor, dolor, rubor and tumor meaning heat,
pain, redness and swelling. These physical observations of inflammation are the effects
of cytokines on local blood flow, which allows the accumulation of fluid and cells in the
inflamed area. Primary mediators of inflammation such as interleukin-1 (IL-1) and tumor
necrosis factor (TNF), which are released from injured tissue, cause dilation of blood
vessels around the area of injury. Endothelial cells lining the blood vessels swell and
partially retract allowing the leakage of fluid into the injured area. The increase in body
temperature is a host response to proinflammatory mediators that decreases viral/bacterial
replication and increases antigen presentation by APCs. Migration and extravasation of
leukocytes depend on interactions with the endothelium. Expression of adhesion
molecules such as selectins and integrins and their interaction with corresponding ligands


82
SPI-2 of RPV shares 92% amino acid homology with CrmA, as shown in Figure
7. However these two proteins differ in the reactive site loop at positions P5 and P6,
thereby not excluding the possibility that the two proteins may have different inhibitory
specificities with respect to activated caspases in apoptotic cells. It has been reported that
there is at least one example of a caspase that recognizes residues away from the PI
cleavage site as far as the P5 residue. Therefore it is possible that the differences
between SPI-2 and CrmA at P5 and P6 may in fact play a role during virus infection.
Published reports from our laboratory have shown that caspases are activated during RPV
but not CPV infections (236). In vitro experiments with purified SPI-2 and CrmA have
indicated that both the proteins could inhibit the activity of caspase-1 with similar
efficiencies. These results suggest that although the two proteins SPI-2 and CrmA
function similarly in vitro, they may not be functionally equivalent in vivo.
Swapping the two genes between the viruses allows us to examine any differences that
might be present between SPI-2 and CrmA. If SPI-2 and CrmA have distinct properties,
then one would expect an infection of LLC-PK1 cells where CrmA is expressed in place
of SPI-2 in RPV to inhibit the induction of apoptosis (unlike wt RPV infected cells). And
similarly SPI-2 expressed in place of CrmA in CPV should induce apoptosis in LLC-PK1
cells (unlike wt CPV infections). However if there were an additional factor unique to
CPV that is required in addition to CrmA to inhibit the induction of apoptosis, then
blockage of apoptotic induction would be observed only during CPV infection regardless
of SPI-2 or CrmA expression. The results of this project are presented in this thesis and
are discussed.


123
A.
Wt CPV
hr p.i 2 6 8 10 12 14 16 18
CrmA
36 kDa -
1 2 3 4 5 6 7 8
B.
hr p.i
SPI-2
36 kDa -
1 2 345 6789
CPVAcrmA::SPI-2
2 6 8 10 12 14 16 18 Mock
Figure 11. Expression of CrmA and SPI-2 during CPV infections. Extracts were
made from CV-1 cells infected at an MOI of 10 harvested at various times post infection,
subjected to SDS-PAGE and immunoblotted with monoclonal antisera to (A) CrmA
expressed in wt CPV and (B) SPI-2 expressed in CPVAcrmA::SPI-2. Arrows indicate the
position of migrating CrmA/SPI-2 proteins at 38 kDa. Immunoblots indicate that the
recombinant proteins are being expressed from during virus infection.


26
of a 20 amino acid leader sequence (67). Maturation of IL-8 occurs extracellularly with a
number of proteases being implicated in the cleavage process. Two forms of mature IL-8
have been observed to be secreted from activated neutrophils IL-8 [77] and IL-8 [72]
containing 77 or 72 residues respectively which can be further processed by neutrophil
elastase, cathepsin G and proteinase-3 to give N-terminal truncated forms that are more
active (302). Neutrophil gelatinase B, a secreted matrix metalloproteinase (MMP-9) has
also been shown to cleave mature IL-8 [77] to more active truncated forms (431). Thus
inflammatory exudates containing proteases can potentiate more active forms of IL-8.
Near sites of production, chemokines can form oligomers on endothelial or
extracellular matrix, thereby forming a gradient with higher concentrations of chemokine
near the inflammatory stimulus (298). In addition to attracting neutrophils, IL-8 has also
been shown to have chemotatic effects on a number of other cells including basophils,
eosinophils, T lymphocytes and macrophages (366). The ability of IL-8 to attract
neutrophils has been disputed by both in vitro and in vivo experiments involving
intradermal injections in humans (218, 222, 405). In addition to being secreted by
neutrophils, IL-8 is also secreted by keratinocytes, fibroblasts, endothelial cells and T
cells (310). IL-8 has been demonstrated to have mitogenic effects (353). Subsequently
constitutive IL-8 expression has been observed in many types of human cancers (366).
IL-8 has also been shown to induce angiogenesis (199).
Although monomeric IL-8 is biologically active, IL-8 can also exist as a dimmer
with the disulphide bonds playing important roles in anchoring the cores of the dimmer
together (70). N-terminal residues Glu4, Leu5 and Arg6 (ELR) have been shown to be
absolutely essential for receptor binding (67, 144). The ELR motif is found to be present


63
apoptosis in Sf cells infected with a AcNPV P35 mutant (80, 337). The CplA? and
OpWV proteins (isolated from baculoviruses Cydia pomonella granulosisvirus and
Orgyia pseudotsugata nucleopolyhedrovirus respectively) can also inhibit apoptosis
induced by a variety of signals including induction by, actinomycin D, expression of the
reaper and Doom genes of Drosophila melanogaster and expression of ICE in a
mammalian system (153). The baculovirus IAPs mode of inhibiting the S/caspase-1
activity is thought to affect the activation of this insect caspase, implying that the
mechanism by which IAPs function is different from the P35 mode of action (365). A
number of IAP homologues have been found in insects and mammals including the
human neuronal apoptosis inhibitory protein (NAIP), cIAP-1, cIAP-2 and XIAP (350).
The mechanism by which viral IAPs act in inhibiting apoptosis is not fully understood.
Studies with the crystal structures of human XIAP has elucidated a possible mechanism
of action that could be similar for all IAPs. The BIR3 domain of XIAP is thought to aid
the cleavage of caspase-9 and directly bind the small subunit at the C-terminus of
caspase-9 inactivating it (383, 390). With respect to caspase-3 and -7, XIAP seems to
have a completely different mode of inhibition than that seen with caspae-9 (60, 164,
338). In this case BIR2 domain is involved and complexes with caspase-3 and -7. A
linker domain N-terminus to BIR2 functions to block the substrate groove in the caspase,
in a backward or opposite orientation that is different from substrate recognition
sequences making it a novel mechanism for caspase inhibition. XIAP is not a substrate
for caspase-3 or -7 but rather functions to mask the active site. It will be of interest to
know whether the viral IAPs also function in a similar manner.


182
CrmA. If the differences between the properties of SPI-2 and CrmA as we have shown
were indeed dependent on the differences within the RCL (notably at P5 and P6), then it
would be of importance to consider these regions when designing therapeutic peptide
inhibitors of caspases (288). Differences as far as the P5 position have been shown to
influence substrate specificity (239). Peptide inhibitors of caspases are typically small
and comprise of residues meant to mimic RCL targeting of enzymes. Caspase-1
inhibitors have shown promise in the treatment of rheumatoid arthritis. Peptide inhibitors
designed for the inhibition of caspases involved in apoptosis are yet to reach the clinical
stage. Most of the work in therapeutics is centered on inhibiting caspase-3, the terminal
apoptotic caspase. In animal models of ischaemia, caspase inhibition improved survival.
Currently available peptide ketone inhibitors of caspases are relatively non-specific for
individual caspase members but do provide evidence for the therapeutic potential in
blocking caspase activity in certain disease models. While designing such molecules it
may be worth considering the pathological context of usage since our experiments show
that minor differences in the P5 and P6 regions of an RCL may be sufficient to modify
the outcome of an infection.
Therapeutic peptides based on targeting apoptotic and inflammatory caspases are
still in their infancy. Nevertheless this is an emerging and promising area of clinical
research as evidenced by various pharmaceutical companies that have an active interest in
the development of these technologies.


GM-CSF
granulocyte-monocyte colony stimulating factor
HPRT
hypoxanthine guanine phosphoribosyltransferase
IAP
inhibitors of apoptosis proteins from baculovirus
ICE
interleukin 1P converting enzyme
IFN
interferon
IL
interleukin
IRAK
IL-1R associated kinase
ITA
inhibitor of T-cell apoptosis
ITR
inverted terminal repeat
JNK
Jun N-terminal kinase
kbp
kilobase pairs
LLC-PK1
pig kidney cell line
MAPK
mitogen activated protein kinase
M-CSF
monocyte colony stimulating factor
MMP
matrix metalloproteinase
MOI
multiplicity of infection
MPA
mycophenolic acid
NADP
nicotinamide adenine dinucleotide phosphate
NF-kB
nuclear factor kappa B
NGF
nerve growth factor
NIK
NF-kB inducing kinase
NK
natural killer cell
NO
nitric oxide
Xll


207
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32 Caspase-3 activity in the presence of chicken extracts 172
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34 CPVAIL-1 PR fails to induce inflammation during CAM infections 175
x


4
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the extracellular milieu and act as microbicidal agents. The release of oxygen radicals
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repaired.


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ROLE OF CrmA IN REGULATING INFLAMMATION AND APOPTOSIS DURING
COWPOX VIRUS INFECTION
By
RAJKUMAR NATHANIEL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003


144
strengthens the hypothesis that CrmA acts as a protease inhibitor to prevent the activation
of caspases.
Recombinant CPV Infections of CAMs
During CPV infections of CAMs, CrmA is presumed to act directly on caspase-1
to prevent the maturation of inflammatory cytokines IL-1P and IL-18 from their
respective precursors. If this hypothesis were true, then replacing CrmA in CPV with
other caspase inhibitors should produce similar results as wt CPV. We chose pan-
caspase inhibitor P35 based on the ability of P35 to inhibit caspase activation in a number
of species (250). Indeed P35 is able to function as caspase inhibitor and as a CrmA
replacement during CPV infections of LLC-PK1 cells, therefore we expected P35 to
functionally replace CrmA in CPV infections of CAMs.
Second, we chose SERP2 as a replacement for CrmA in CPV to answer the
question of whether poxvirus serpins from different poxviruses had similar functional
targets. If this were true, we would expect SERP2 to functionally replace CrmA in CPV.
Considering that SERP2 failed to inhibit caspase induction within the context of CPV
infections of swine cells, we were unsure of what to expect during CAM infections using
the various CPV derivatives.
Third, the ability of CrmA to inhibit an inflammatory response during CPV
infections of CAMs may not be related to CrmAs ability to act as a protease inhibitor,
more specifically a caspase inhibitor. The CrmA D303A mutation in the RCL alters
target protease specificity. Indeed CPVCrmA D303A is unable to block the activation of
caspases in pig cells and therefore is an excellent candidate to answer the last hypothesis.


160
assumption that the N-terminal portion of chicken proIL-1 P, like mammalian proIL-ip,
was removed by processing during the activation process. These observations would be
consistent either with an alternative caspase-independent mechanism of processing, or a
somewhat different cleavage recognition site for avian caspase-1. However, in the case
of proIL-18, the vertebrate cleavage site is completely conserved within the known
chicken proIL-18 sequence (354). Given the known proinflammatory functions of IL-1 (3
and IL-18, we sought to determine whether any proIL-ip or proIL-18 processing activity
was detected within infected CAMs.
Despite the absence of the two critical aspartic acid residues, we first tested the
ability of infected CAM extracts to process 35S labeled chicken proIL-ip (Fig. 27).
Lysates were prepared from infected membranes and incubated with 35S radiolabeled
chicken proIL-1 p. Processing activity was measured by monitoring the cleavage of the
precursor molecules with SDS-PAGE and subsequent autoradiography. Precursor
chicken IL-1P migrated at approximately 29 kDa. The recombinant human caspase-1
(1U), as expected, based on the sequences (446), failed to process the chicken precursor
(Fig. 27, lane 1). More importantly, we also failed to see any processing of chicken
proIL-1 p by any of the infected CAM extracts (Fig. 27, lanes 2-6). This result suggested
two possibilities. First, the possibility that chicken IL-1 P is not involved in the
inflammation on CAMs. Second possibility is that if chicken IL-1 p were to be involved
in the inflammation on CAMs, the chicken IL-1P precursor itself is biologically active.


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81
the intra dermal route with vaccinia virus deleted for SPI-2, showed enhanced pathology
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RPV SPI-2 and CPV CrmA Equivalency
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the fact that RPV encodes a functional SPI-2 (CrmA) protein.
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of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity


CHAPTER 4
DISCUSSION
Equivalency of SPI-2 and CrmA
CrmA was the first viral serpin to be characterized (316). All Orthopoxviruses
analyzed to date have been shown to contain the CrmA homolog SPI-2 (250). Overall
SPI-2 and CrmA share >90% identity and >92% similarity (250). Ectopic expression of
CrmA or SPI-2 can prevent apoptosis induced by TNF (407, 425). In addition both
CrmA and SPI-2 can inhibit caspase-1 with similar efficiencies (236). But despite their
high degrees of identity, published reports have shown that SPI-2 and CrmA may not
function similarly during virus infection. While CrmA can control inflammation in the
CAM model of infection, SPI-2 in vaccinia virus is unable to function in a similar manner
(304, 316). Previous reports have shown that during pig kidney cell infections, CrmA
functions to control apoptosis whereas RPV containing the functional CrmA homolog
SPI-2, is unable to prevent the induction of apoptosis (236). In the intranasal model of
infection, deletion of CrmA from CPV causes mild attenuation (one order of magnitude
in LD50 levels) whereas deleting SPI-2 from vaccinia virus has no effect on infection
(193,412).
In this study we attempted to determine the equivalency of the SPI-2 and CrmA
by switching the coding regions of the two genes between the two viruses RPV and CPV.
Swapping the entire ORFs between the two viruses allows us also to examine the effect,
if any, the individual viral genomes may have on the expression and function of SPI-
178


103
Construction of Recombinant CPV Expressing P35
The P7.5-gpt cassette which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the Sail and Apal sites of pBS-CCF
resulting in the shuttle vector pBS-CgS/A.
The Pn-lacZ cassette which allows for blue-white screening with X-Gal (5-bromo-4-
chloro-3-indolyl-P-D-galactopyranoside) was PCR amplified from pSCl 1 (61) with Vent
polymerase using primers FS 129 (5-
GTCAGATCCATGGTTGAATTCCGAGCTTGGCTG-3 ) containing the Ncol site and
FS 130 (5 AGTC AACGCCCGGGT ACGCT C AC AG AATTCCCG-3 ) containing the
Smal site. The lacZ PCR product was cloned into the Ncol and Smal sites of pBS-
CgS/A resulting in pCglacZ. Using pCglacZ the CrmA open reading frame in CPV was
replaced with lacZ by transient dominant selection (97) to make CPVAcrmA::lacZ.
The P35 gene from the baculovirus Autographa californica NPV was provided by
Lois Miller (University of Georgia, Athens). The plasmid pC35 was constructed by
inserting the P35 coding region between the Ncol and Smal sites of pBS-CgS/A by
recombinant PCR (421) such that the start codon of P35 exactly replaces the ATG at the
Ncol site. The primer pair used to generate the left flank was FS 1 (5-
GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-3 ) containing
an Xbal site (underlined) and FS 91 (5 -GGCAATCGATTTTGTTG-3 ). The primer pair
used to generate P35 was FS 89 (5-
CAACAAAATCGATTGCCATGTGTGTAATTTTTCCGG-3 1 (underlined portion is
complimentary to FS 91) and FS 74(5-
T ACGT C ACCCGGGTT ATTT AATT GT GTTTA AT ATT AC-3 ) containing a Smal site


61
TNFRI and Fas induced apoptosis (32). MCI59 of Molluscum contagiosm virus contains
two DEDs, binds both caspase-8 and FADD and blocks apoptosis induced by Fas, TNF
and TRAIL (120). Similar vFLIPs have been found in a number of herpes viruses some
of which associate with kinases of the IKB kinase complex (127, 229).
Baculovirus Caspase Inhibitor: P35
The baculovirus anti-apoptotic P35 proteins are unique in that they do not share
homologies with any other known proteins. Interestingly these proteins share no
homology to poxvirus serpins, yet they inhibit similar target proteinases. P35 was first
identified in the insect virus Autographa californica nuclear polyhedrosis virus (AcNPV)
where P35 mutants induce apoptosis in the Sf-21 insect cell line (isolated from
Spodoptera frugipedra) (68). P35 proteins from a number of baculoviruses show distinct
abilities to inhibit apoptosis (267). Most of the P35 functional data generated to date
have used ytcNPV P35. In vitro studies have implicated P35 to be a suicide substrate for
caspases (4). P35 has been shown to inhibit all three groups of mammalian caspases and
specifically caspases-1, -3, -6, -7, -8 and -10 (474). In addition P35 has also been shown
to inhibit the C.elgans CED-3, Sf caspase-1 as well as gingipain (4, 378, 459). Recently,
the crystal structures of P35 with caspase-8 an apical initiator caspase and also with
effector Sf caspase-1 have revealed clues to its function mechanisms (92, 106, 458). The
reactive site loop (RCL) of P35 associates with the molecules main p sheet core such
that the Asp residue required for protease recognition is exposed at the apex of the loop.
Following cleavage of the RCL, there forms a covalent thio-ester linkage between the
target proteinase and P35. Cleavage also induces a dramatic conformational change in


62
P35 where the N-terminal of P35 gets repositioned into the active site of the caspase
protecting the thio-ester linkage from hydrolysis. Interestingly the presence of certain
motifs in effector caspases but absent in initiator caspases explains the preference of P35
to complex with terminal rather than apical caspases. Ectopic expression of P35 has
shown it inhibits apoptosis in a number of systems including insects, nematodes and
mammalian cells where the apoptotic signals tested included virus infection, induction by
actinomycin D, induction by Fas and TNF, NGF depravation, developmental^
programmed cell death and induction by X-radiation (259). In each case, P35 was able to
either block completely or significantly delay apoptosis. In the light of these studies, the
broad range of activity ascribed to P35 is suggestive that the molecule is a pan-caspase
inhibitor interrupting the activity of a highly conserved and ubiquitous component of the
death machinery.
Baculovirus Inhibitor of Apoptosis Proteins (IAP)
The family of inhibitor of apoptosis (IAP) was first described in baculoviruses by
Lois Miller and others (75). IAPs have also been identified in yeast, worms, insects and
mammals (350). Two zinc-binding motifs are characteristic of IAPs, the first is the
baculovirus IAP repeat (BIR) and the second being a RING domain. The BIR motif can
be found in multiple copies in a given IAP, while the RING domain is typically a single
copy located at the carboxy terminal end of the molecule (350). The RING domains
function to catalyze the degradation of IAPs and target protein through ubiquitylation
processes. The BIR domains seem to be essential for IAPs to function as anti-apoptotic
agents (89). Baculovirus IAPs were discovered by virtue of their ability to block


142
A. Mock
B. CPV
C. CPVAcrmA::SERP2 D. CPVCrmA D303A
Figure 19. Morphological changes in LLC-PK1 cells infected with
CPVAcrmA::SERP2 or CPVCrmA D303A. Pig kidney cells were infected at MOI 10
and stained with DAPI at 16 hours post infection. Panels indicate the different viruses
used for infection (A) Mock, (B) CPV, (C) CPVAcrmA::SERP2 and (D) CPVCrmA
D303A. Examples of apoptotic bodies, characteristic of cells undergoing apoptosis are
indicated by arrowheads.


29
short, IL-8 is particularly important in the innate immune response by directing
neutrophil migration to sites of infection.
Avian Cytokines
A number of homologs for mammalian proinflammatory cytokines have been
identified in birds. A review of avian and mammalian cytokines reveals that even though
they function similarly, some of their primary structures seem to differ significantly.
Avian homologs of TNF-a, IL-1 (3, IL-18 and a number of CXC chemokines have been
identified so far. A majority of these molecules were discovered in chickens, in addition
to duck, turkey, pheasant, quail and guinea fowl (384).
TNF-a
TNF-a-like activity can be detected in chick embryos as early as day 1 (452).
The pattern of expression throughout the growing embryo suggested a role for TNF in
apoptosis during differentiation of the neuronal system as well as in tissue remodeling.
Indeed cells undergoing DNA fragmentation associated with apoptosis were
demonstrated to be closely associated with TNF-a immunoreactivity (451). Chicken
macrophages stimulated with bacterial LPS or the intracellular protozoan Eimeria,
secreted a TNF-like activity that was preferentially cytotoxic to chicken fibroblasts
indicating its role in bacterial and coccidial infections (471). Partial purification of
stimulated macrophage supernatant fractions containing TNF-like activity revealed a 17
kDa protein that cross reacted with antibodies to human TNF-a (329). Although the
partially purified protein induced biological activities related to TNF-a in chicken


34
bind both TNF-a and (3 and block their cytolytic activity in vitro (233). In addition to
being present in cowpoxvirus, CrmE is also present in other poxviruses such as vaccinia
virus (352). The cowpox CrmE was also found to only inhibit TNF-a activity. More
recent work has shown the vaccinia CrmE protein to also bind TNF-a but not (3 (333). In
this study CrmE was found to inhibit the cytotoxic activities of human TNF-a but not
those of mouse or rat. Vaccinia strain USSR deleted for CrmE was attenuated in an
intranasal mouse model of infection. But vaccinia WR strain, engineered to either
express the USSR CrmE, or CPV CrmB or CPV CrmC or CPV CrmE genes displayed
enhanced virulence indicating the influence of viral TNFRs on disease outcome. The
CD30 molecule is a member of the TNF receptor family and is associated with Th2
responses although its exact function role is unclear at present (31). The important role
of CD30 in viral infection is evidenced by the fact that cowpoxvirus encodes a secreted
CD30 homologue. Another virus that interferes with TNF is the herpes human
cytomegalovirus that encodes UL144, which also belongs to the TNFR family (30).
While the ligand that binds UL144 is yet to be identified, its importance in viral infection
is demonstrated by the presence of antibodies to UL144 in patient sera.
IL-1 Inhibitors
Members of poxviruses also encode molecules that interfere with IL-1 (3 either in
its production or signaling. The subject of this dissertation work is the cowpoxvirus
CrmA protein that will be discussed in more detail later. Briefly, CrmA is believed to
inhibit the processing of proIL-1P by blocking the activity of the processing enzyme,
caspase-1 (331). The importance ofIL-1p involvement in viral infection is further


125
A. Mock B. RPV
C. RPVASPI-2::lacZ D. RPVASPI-2::crmA
E. RPVASPI-2::P35
Figure 12. Morphological characteristics of LLC-PK1 cells infected with RPV
derivatives. Pig kidney cells were infected at MOI 10 and stained with DAPI at 16 hours
post infection. Panels indicate the different viruses used for infection (A) Mock, (B)
RPV, (C) RPVASPI-2::lacZ, (D) RPVASPI-2::crmA and (E) RPVASPI-2::P35.
Examples of apoptotic bodies, characteristic of cells undergoing apoptosis are indicated
by arrowheads.


72
neurotrophic factors
PEDF (SERPINFI)
neuroserpin (SER PI Nil)
PNI (SERPINEI)
tumor cell invasion
prohormone conversion
renal development
ACT (SERPINAS)
megsin (SERPINB7)
sperm development-
PCI (SERPINAS)
coagulation
hormone transport
9
Q
9
r!cis!S8i h PCI (SERRINAS)
inflammation &
T complement activation
UIPI (SERPINAl)
alA CT (SERPINA3)
KAL (SERPINA4)
UNE! (SERPINBI)
t i ov-serpius (SERPINB*)
blood pressure regulation
AGT (SERPINAS)
7~ angiogenesis
PEDF (SERPINF I)
maspin (SERP1NB5)?
ATIII (SERPINCI)?
PAH (SERPINEI)?
T
PCI (SERPINASj
ATIII (SERPINCI)
HCFII (SERPINDI)
PAH (SERPINEI)
ECM maintenance
and remodelling
alPI (SERPINAl) PNI (SERPINE2)
OlACT (SERPINA3) HSP47 (SERPINHI)
PAll (SERPINEI) CBP2 (SERPINHI)
B cell development
Q^ v
centerin (SERPINA9) %
fibrinolysis
T
PAI2 (SERPINB2)
PAll (SERPINEI)
02AP (SERPINP2)
apoptosis
I alPI (SERPINAl)
aJACT (SERPINA3)
ov-serpms (SERPINB*)?
microbial infection
Cl Inh (SERPINGI)
ov-serpins (SERPINB*)?
Figure 4. Serpins as physiological regulators. Serpins are involved in numerous
functions both as positive and negative regulators. From reference 368.


174
A.
CPV Genome
1
vIL-ipR
194007 194987
\/
224,501 bp
1 2
Figure 33. Construction of CPV AIL-1 PR. (A) The location of vIL-1 PR within the
CPV genome is indicated. The left and right flanking regions (hatched boxes) within
CPV vIL-ipR ORF were PCR amplified and cloned into pBluescript II KS+. The
E.coli.gpl gene was PCR amplified and cloned within CPV IL-1R left and right flanks
resulting in pBS-CPV-IL-IRKO. Insertional mutant virus CPV AIL-1 PR (KO) virus was
generated by homologous recombination and selection for MPA resistant (96) virus. (B)
PCR products were generated from CPVAIL-ipR viral DNA purified after 5 rounds of
plaque purification. A PCR product migrating at approximately 1.2 kbp represents the
recombinant KO viral construct (lane 1). A viral DNA sample prepared during the initial
stages of plaque purification show the presence of an additional 1.0 kbp PCR product
(lane 2) representing vIL-ipR in wt CPV that would be present along with the
recombinant virus in plaques, (see Recombinant Virus Construction in Materials and
Methods for detailed description of virus construct).


204
91. Eckhart, L., W. Declercq, J. Ban, M. Rend, B. Lengauer, C. Mayer, S.
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caspase-14 activation. J. Invest Dermatol. 115:1148-1151.
92. Eddins, M. J., D. Lemongello, P. D. Friesen, and A. J. Fisher. 2002.
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188
failed to detect mature IL-1 (3 under any CAM infection condition and also found that P35
and SERP2 function like CrmA in blocking caspase-1 (when added exogenously)
mediated processing of IL-ip (Fig. 28). This suggested that during CPV infection if
caspase-1 were to be involved, P35, SERP2 and CrmA could block it.
These results suggested that chicken IL-1 (3 is not involved in the inflammation
during CPV infection of CAMs. Alternatively if chicken IL-1P is responsible for the
inflammation on CAMs, chicken proIL-1P is possibly active as a precursor. We would
not be able to test pocks for proIL-1 p production since all the inflammatory pocks
contained high levels of heterophils, which are known producers of IL-ip.
Because of the ambiguity surrounding activation of avian IL-1 p, we chose to
investigate the ability of infected CAMs to instead cleave proIL-18 to active IL-18 as an
indicator of caspase-1 activity. The chicken proIL-18 sequence contains the conserved
Asp29 residue required for caspase-1 mediated cleavage (354). Therefore it was not
surprising that CPVAcrmA::lacZ and CPVCrmA D303A extracts from CAMs that were
known to contain caspase activity (Fig. 26), were able to process chicken proIL-18 (Fig.
29). Despite reports of caspase-3 being able to process proIL-18 (122, 133), we found
little, if any, processing of chicken proIL-18 by up to 15 U of recombinant human
caspase-3 (Fig. 30). The inability of human caspase-3 to process chicken proIL-18 could
be due to species specificity, i.e. a low affinity of human caspase-3 for the chicken
substrate. However, we have also shown that the chicken proIL-18 processing ability by
CPVAcrmA::lacZ infected CAM extracts could be inhibited by pre-incubating extracts
with Ac-DEVD-CHO, a caspase-3 inhibitor or Z-VAD-FMK, a pan-caspase inhibitor but
not Ac-WEHD-CHO, a caspase-1 inhibitor (Fig. 31). Therefore we conclude that the


20
activation (190). Thus is seems that TNF-RI signaling of NF-kB is mediated via RIP
while TRAF-2 enables JNK activation.
TNF-RII is a 75 kDa receptor that is co-expressed with TNF-RI predominantly in
endothelial and blood cells and is involved in passing bound TNF to TNF-RI for
signaling (151, 403). In addition, TNF-RII can directly signal NF-kB activation (152).
TNF-RII has been found to associate with a number of proteins including TRAF-1,
TRAF-2, cellular inhibitor of apoptosis protein (cIAP) -1 and cIAP-2 (343). While
TRAF-1 and -2 can form heterodimers, only TRAF-2 contacts the receptor directly (345).
Similarly cIAP-1 and -2 interact indirectly with the receptor by associating with TRAF-1
and -2 via their N-terminal BIR (baculovirus IAP repeat) domain (343). Cellular IAPs
have been shown to inhibit the induction of apoptosis by interacting with caspases-3 and -
7 directly as well as target these proteases to degradation by ubiquitination (163, 346).
Cellular IAPs have also been reported to direct the ubiquitination of TRAF-2 (227). It is
unclear at present if cIAPs have any role in the activation of NF-kB directly. Expression
of TRAF-2 is sufficient to induce NF-kB and JNK activation under certain conditions
(232, 344).
A small subset of TNF binding proteins are the soluble TNF receptors that
represent the extracellular cytokine binding domains that have been shed from the cell
surface by TACE mediated proteolytic cleavage (76, 379). The soluble receptors function
to neutralize the biological activity of TNF by antagonizing the binding of cellular
receptors (94, 179, 364, 379, 406). The outcome of TNF activity depends on the
composition of the signaling complex and can either lead to the induction of apoptosis or
activation of JNK and NF-kB pathways that function to mediate cell survival (232).


107
Table 5. List of plasmids constructed
Plasmid
Properties
1. pBS-RLF
SPI-2 left flank in pBlueScript Xbal and EcoRI
2. pBS-RCF
SPI-2 right flank in pBS-RLF EcoRI and Hindlll
3. pBS-Rgpt
Eco.gpt in pBS-RCF EcoRI
4. pRSG
CrmA in pBS-RCF Ncol and Smal
5. pBS-RgS/A
Eco.gpt in pBS-RCF Sail and Apal
6. pRglacZ
LacZ in pBS-RgS/A Ncol and Smal
7. pR35
P35 in pBS-RgS/A by recombinant PCR
8. pBS-CLF
CrmA left flank in pBlueScript Xbal and EcoRI
9. pBS-CCF
CrmA right flank in pBS-CLF EcoRI and Hindlll
10.pBS-Cgpt
Eco.gpt in pBS-CCF EcoRI
ll.pCSG
SPI-2 in pBS-CCF Ncol and Smal
12.pBS-CgS/A
Eco.gpt in pBS-CCF Sail and Apal
13.pCglacZ
LacZ in pBS-CgS/A Ncol and Smal
14.pC35
P35 in pBS-CgS/A by recombinant PCR
15.pCSERP2
SERP2 in pBS-CgS/A Ncol and Smal
1 .pAlterEX-1 CrmA
CrmA in pAlterEX-1 EcoRI and Hindlll
17.pAlterEx-1D303 ACrmA
CrmA D303A generated by mutagenesis
18.pBS-D303ACgS/A
CrmA D303A in pBS-CgS/A
19.pBS-CPVIL-1R-LF
CPVvIL-ipR left flank in pBluescript EcoRI and PstI
20.pBS-CPVIL-1R-LF-MPA
Eco.gpt in pBS-CPVIL-1 R-LF PstI and Smal
21 .pBS-CPVIL-1RKO
CPVvIL-lpR right flank in pBS-CPVIL-1 R-LF-MPA


208
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138
illustrated in Figure 9. It is important to note that in each construct, the native CrmA
promoter regulated the relevant gene, CrmA D303A, P35 or SERP2.
The expression of the various proteins from the CrmA promoter of the
recombinant CPVs was analyzed by immunoblots of infected cell lysates (Fig. 18). The
data show expression of SERP2 (Fig. 184A) or CrmA D303A (Fig. 18B). SERP2 within
the context of CPV migrates at 34 kDa as expected (Fig. 184A, lane 1). In Fig. 184B,
lane 5, we note that CrmA from wild type CPV infections migrates as a 38 kDa protein,
whereas the CrmA D303A protein has a somewhat slower mobility resulting in a band
migrating at approximately 40 kDa (Fig.l8B, lane 7). We have noted altered
electrophoretic mobilities of serpins through mutations within the PI RCL mutations
previously for SPI-1 (Kristin Moon and Richard Moyer unpublished data) and SERP2
(Peter Turner and Richard Moyer unpublished data). The absence of CrmA
immunoreactive band in CPVAcrmA::lacZ (Fig. 18B, lane 6) authenticates this virus
construct. Each of the proteins analyzed appeared to be quite stable within the context of
CPV infected cells (data not shown).
Infections of LLC-PK1 Cells with CPV Recombinants: DAPI Stained Cells
It has been demonstrated that CrmA functions to inhibit caspase activation during
CPV infections of LLC-PK1 cells and thus prevents apoptotic induction in these cells.
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establish CrmAs role during CPV infection, we first wanted to determine the outcome of
pig kidney cell infections in terms of caspase induction by the various recombinant
CPVs. We have already observed that P35 replacements of CrmA in CPV were able to


224
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Stereochemistry, total synthesis, and biological activity of 14,15-dihydroxy-
5,8,10,12-eicosatetraenoic acid. J. Biol. Chem. 259:13011-13016.
324. Rahmani, Z., K. W. Huh, R. Lasher, and A. Siddiqui. 2000. Hepatitis B virus
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2846.


100
pBluescript II KS(+) (Stratagene) resulting in pBS-RLF. The downstream flanking
sequence of the SPI-2 coding region was PCR amplified with from RPV genomic DNA
using primers GM 20 (5-
ATCG AT CGAATTCCCGGGC AT AT GCC ATTTTTTTT A A A A AA AAT AG A AAA A A
CATG-3) containing EcoRI, and Smal sites (underlined) and GM 22 (5-
TGCTACAAGCTTGATGAACACTGATTCCGCATC-3) containing a Hindlll site
(underlined). The right flank PCR product was cloned into the EcoRI and Hindlll sites of
pBS-RLF resulting in pBS-RCF.
The P7 5-gpt cassette that confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the EcoRI site of pBS-RCF resulting in
the shuttle vector pBS-Rgpt. RPVASPI-2::Eco.gpt was created using pBS-Rgpt and
wild type (wt) RPV by homologous recombination and selection of recombinant virus on
medium containing mycophenolic acid. The CrmA gene from pTMIHisCrmA (Peter
Turner unpublished) was re-cloned into the Ncol and Smal sites of pBS-RCF to generate
pRSG. The recombinant RPVASPI-2::CrmA virus was created by reverse selection on
STO cells using 6-thioguanine (170) using pRSG and RPVASPI-2::Eco.gpt.
Construction of Recombinant RPV Expressing P35
The P7 5-gpt cassette which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and re-cloned into the Sail and Apal sites of pBS-RCF
resulting in the shuttle vector pBS-RgS/A.
The Pn-lacZ cassette which allows for blue-white screening with X-Gal (5-bromo-4-
chloro-3-indolyl-(3-D-galactopyranoside) was PCR amplified from pSCl 1 (61) with Vent


126
A. Mock
B. CPV
D. CPVAcrmA::SPI-2
, ^ ****
¡ft 9
**
* m
E. CPVAcrmA::P35
mm
. 5*
A 9
m
j
Figure 13. Morphological characteristics of LLC-PK1 cells infected with CPV
derivatives. Pig kidney cells were infected at MOI 10 and stained with DAPI at 16 hours
post infection. Panels indicate the different viruses used for infection (A) Mock, (B)
CPV, (C) CPVAcrmA::lacZ, (D) CPVAcrmA::SPI-2 and (E) CPVAcrmA::P35.
Examples of apoptotic bodies, characteristic of cells undergoing apoptosis are indicated
by arrowheads.


180
(>92%), their identical properties in vitro, the presence of similar but not identical
residues in the P5 and P6 positions RCL, the differences between the two molecules are
highlighted depending on the viral context of expression.
There are at least two major differing regions when comparing CrmA and SPI-2
molecules as seen in Figure 7. Overall, the CPV CrmA and RPV SPI-2 proteins are
>92% identical and within the RCL differ only at positions P5 and P6 (236). The second
region is within the viral serpin back-bone or region outside the RCL. Structural
comparison of CrmA with the prototypical serpin alpha-1 anti-trypsin reveals the absence
of a region termed helix D within CrmA (250). The helix D region is seen to function as
binding region for co-factors that modulate serpin function. Within CrmA, although
helix D is missing, it is replaced by the addition of a novel p-strand designated Sla
between residues 50 and 60. Interestingly RPV SPI-2 and CPV CrmA also differ in the
Sla region. It is therefore probable that differences seen in the function of CrmA and
SPI-2 are due to minor differences within the RCL and/or within the Sla region.
To differentiate between these two possibilities, it would be necessary to engineer
changes in SPI-2. First, the P5 and P6 residues in SPI-2 RCL could be mutated to mimic
CrmA RCL and see whether these changes have any effect on SPI-2 function within the
context of RPV infections. Second, the Sla region in SPI-2 could be made to look
exactly similar to the corresponding region in CrmA and the effect on SPI-2 function
within RPV context could be addressed. There lies the possibility that the differences
seen between SPI-2 and CrmA function within RPV are due to differences not associated
with either the RCL or Sla regions.


9
family, which includes in addition IL-la and IL-1 receptor antagonist (IL-IRa) (98).
Both IL-la and IL-ip are synthesized as 31 kDa precursors that are processed to 17 kDa
mature cytokines by cysteine proteases calpain and caspase-1 respectively (55, 58, 189,
238, 413). ProIL-la is processed between residues 112 to 113 by calpain whereas proIL-
1 p is cleaved by caspase-1 first at Asp27 followed by cleavage at Aspl 16 to yield mature
cytokine (198, 395). ProIL-la also contains a nuclear localization signal at residues 79-
86 (448). Interestingly while proIL-1 p has an absolute requirement for processing to be
functional, proIL-la is biologically active as a precursor (58, 253, 270). Neither IL-la
nor *p contains signal sequence regions that would target these proteins to the secretory
pathway (99). The predominant soluble form seems to be IL-1 p, whereas IL-la is
thought to be cell associated (21). IL-Ra is a soluble protein that is able to bind IL-1
receptors but is unable to transmit a signal and is thought to control IL-1 mediated
inflammation (86).
There are two receptors identified for IL-1. The 80 kDa IL-1 receptor type I (IL-
1RI) is responsible for signal transduction (Figure 1), whereas the 60 kDa type II receptor
(IL-1RII) does not deliver any signal and therefore functions to regulate IL-1 activity by
acting as a sink (370). The IL-1RI shares 45% homology with the cytoplasmic region of
Drosophila Toll protein (121). In addition to the TIR domain, IL-1RI and II contain
three extracellular immunoglobulin domains. Initial binding of IL-1 P to the receptor is
with low affinity. Crystal structures of IL-1 RI have revealed that binding of IL-1 p to the
receptor induces a conformational change in the receptor that allows the third Ig domain
on the receptor to wrap around the ligand (357, 435). This conformational change in the


184
phenomenon of P35 availability during RPV infections is related to events that occur
during/after viral DNA replication (Fig. 17). Interestingly the induction of caspase
activity also coincides with the timing of viral DNA replication and can be blocked by
AraC (data not shown). Whether P35 stability in RPV is related to caspase induction or
to some other late viral event is yet to be resolved.
It is possible that in RPV infections, P35 is processed/degraded by either a viral or
cellular protease that is activated after 8 hours of infection. Another possibility is that
P35 is inherently unstable but in CPV is rendered stable by another viral factor. We have
also noticed that at MOI of 15 or more, even within the context of CPV, P35 is unable to
completely block the induction of caspase activity in infections of pig cells (data not
shown). Therefore it seems probable that the stability of P35 may depend on events
associated with the virus infection, and this state is exacerbated in RPV but not CPV;
where P35 is able to function as a caspase inhibitor at MOI of 10. Since P35 expressed
normally in CPV is able to function as a caspase inhibitor during virus infection, we
continued to use this recombinant virus in our in vivo studies using the CAM model of
infection.
Function of CrmA during CPV Infections of CAMs
Previous reports of infected CAMs have dealt with lesion morphology and
infiltrating cell types. In this study using the CAM model of infection, we investigate the
biochemical properties of CrmA in order to gain a better understanding of its role during
CPV infection. Consistent with the ability of CrmA to inhibit the inflammatory response
during CPV infections of CAMs, it has been shown that CrmA can directly inhibit


155
infections induce terminal caspase activity to similar levels (Fig. 26). This induction of
terminal caspase activity by infection with CPVCrmA D303A reaffirms that the PI Asp of
the RCL is an essential feature of CrmA function and is required to prevent caspase
induction. Both P35 and SERP2, like CrmA, in the context of CPV, block induction of
terminal caspase activity within pocks (Fig. 26).
The ability of CPVAcrmA::SERP2 to prevent the induction of avian caspases in
both pock and CEF extracts contrasts the results we obtained with similar infections of
swine cells (Fig. 20). These results suggest that the restoration of pock titers seen in both
P35 and SERP2 recombinant infections is due to preventing apoptosis. These results also
imply that the reduction in virus yields seen in CPVAcrmA::lacZ and CPVCrmA D303A
infections were due to a failure to prevent apoptosis and not due to containment by
inflammation and/or the presence of activated heterophils. Thus CrmA appears to control
both inflammation and apoptosis within CAMs and P35 and SERP2 can substitute for
CrmA only to control apoptosis but not inflammation. Since all three proteins act to
inhibit caspases, the possibility exists that CrmA inhibits inflammation by a novel non-
caspase dependent mechanism.
Effect of Caspase Inhibitors on Protease Activity Present in Infected CAMs
Based on the ability to cleave Ac-DEVD-AMC, CAM extracts from
CPVAcrmA::lacZ and CPVCrmA D303A contain a putative caspase-3-like activity (Fig.
26). We then determined the range of caspase activity within CAMs with the use of
caspase specific peptide inhibitors. The peptide Ac-WEHD-CEIO is an inhibitor specific
for human caspase-1, whereas Ac-DEVD-CHO is a potent peptide inhibitor for human


181
Of interest however, is the question of whether the differences seen between SPI-
2 and CrmA have any consequences for RPV or CPV infections in vivo. While there was
no significant difference between the LD50 of CPV and RPV in Balb/c mice, clearly there
are differences between the pathology caused by CPV and RPV infections (412). CPV
infection of mice was always accompanied by severe pulmonary hemorrhage as opposed
to RPV infections that did not caused pulmonary hemorrhage at any time during
infection. RPV infections caused viremia, which was absent from CPV infections.
RPVASPI-2 infected mice showed more extensive infiltrate of inflammatory cells in the
lungs of infected mice when compared with RPV infected mice indicating that SPI-2
played a role in limiting pulmonary inflammation. However, in the case of CPV
infections, CPVAcrmA showed decreased pulmonary pathology and inflammation
compared with CPV infected mice. It has been postulated that the pathology associated
with CPV infections is due to necrosis, which could explain the increased inflammatory
cell response as opposed to CPVAcrmA infections, which probably causes apoptosis in
infected cells, and therefore show decrease in pulmonary inflammation.
The availability of RPV and CPV expressing CrmA and SPI-2 respectively could
be used to study the effects of these genes on pulmonary pathology associated with virus
infection of mice.
Potential Applications
Our results highlight the fact that although the orthopoxviruses RPV and CPV are
considered to be very similar, they behave quite differently during infections of swine
cells and at least RPV is able to differentiate the minor differences between SPI-2 and


68
Figure 3. Poxvirus replication cycle. The cytoplasmic infection of a cell with an
infectious poxvirus particle. Abbreviations: EEV, extracellular enveloped virus; IMV,
intracellular mature virus; IEV, intracellular enveloped virus.


238
468. Yuan, J., S. Shaham, S. Ledoux, H. M. Ellis, and H. R. Horvitz. 1993. The C.
elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-
1 beta-converting enzyme. Cell 75:641-652.
469. Zhang, J., X. Liu, D. C. Scherer, L. van Kaer, X. Wang, and M. Xu. 1998.
Resistance to DNA fragmentation and chromatin condensation in mice lacking the
DNA fragmentation factor 45. Proc. Natl. Acad. Sci. U. S. A 95:12480-12485.
470. Zhang, J. K., D. Cado, A. Chen, N. H. Kabra, and A. Winoto. 1998. Fas-
mediated apoptosis and activation-induced T-cell proliferation are defective in
mice lacking FADD/Mortl. Nature 392:296-300.
471. Zhang, S., H. S. Lillehoj, and M. D. Ruff. 1995. Chicken tumor necrosis-like
factor. I. In vitro production by macrophages stimulated with Eimeria tenella or
bacterial lipopolysaccharide. Poult. Sci. 74:1304-1310.
472. Zheng, H., D. Fletcher, W. Kozak, M. Jiang, K. J. Hofmann, C. A. Conn, D.
Soszynski, C. Grabiec, M. E. Trumbauer, and A. Shaw. 1995. Resistance to
fever induction and impaired acute-phase response in interleukin-1 beta-deficient
mice. Immunity 3:9-19.
473. Zhou, B. B., H. Li, J. Yuan, and M. W. Kirschner. 1998. Caspase-dependent
activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat
cells. Proc. Natl. Acad. Sci. U. S. A 95:6785-6790.
474. Zhou, Q., J. F. Krebs, S. J. Snipas, A. Price, E. S. Alnemri, K. J. Tomaselli,
and G. S. Salvesen. 1998. Interaction of the baculovirus anti-apoptotic protein
p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35
complex. Biochemistry 37:10757-10765.
475. Zou, J., S. Bird, R. Minter, J. Horton, C. Cunningham, and C. J. Secombes.
2000. Molecular cloning of the gene for interleukin-1 beta from Xenopus laevis
and analysis of expression in vivo and in vitro. Immunogenetics 51:332-338.


41
Apoptosis can be induced by a number of stimuli such as radiation, growth factor
depravation, receptor-mediated signaling and viral infections. There are basic pathways
by which a cell can be signaled to initiate apoptosis as shown in Figure 2. One pathway
is extrinsic, mediated via receptor-ligand interactions specifically the receptors related to
the tumor necrosis factor (TNF) and nerve growth factor (NGF) families including
TNFR1, TNFR2 and CD95/APO-l/FasR (147). These specialized cell surface receptors
are termed death receptors (DR) (147). Ligand-receptor interaction results in receptor
oligomerization that causes specific cytoplasmic proteins to be recruited to the receptor
complexes. These cytoplasmic adapter proteins such as TRADD and FADD, then
interact with a group of cellular proteases called caspases. The activation of caspases
leads to the breakdown of cellular homeostasis that culminates in cell death.
Alternatively the second or intrinsic signaling pathway involves mitochondrial
dysfunction (Fig. 2). External cues such as loss of growth factor signals converge with
internal cues such as DNA damage and trigger the permeablization of the outer
mitochondrial membrane and leakage of mitochondrial pro-apoptotic effectors into the
cytoplasm (147). In addition the cytotoxic granule, granzyme B, a serine protease
produced by CTLs and natural killer (NK) cells has been shown to directly activate
caspases as well as the cytoplasmic protein Bid which activates the mitochondrial
pathway (26, 184). The pro-apoptotic stimulus results in the release of an array of
molecules from mitochondrial membranes, which ultimately cause cellular demise that
can be dependent or independent of caspase activation. The activation of caspases is by
far the main biochemical incident responsible for programmed cell death. Thus signaling
through DRs or the disruption of mitochondrial integrity results in the induction of


169
1 2 3 4 5
6 7 8 9 10
Figure 31. Chicken proIL-18 processing activity in CPVAcrmA::lacZ is inhibited by
caspase specific peptides. 35S labeled chicken proIL-18 synthesized in vitro was
incubated with either buffer (lane 1), 200 pg of CPVAcrmA::lacZ extracts from
confluently infected CAMs harvested 48 hours post infection (lane 2), CPVAcrmA::lacZ
extracts preincubated with 100 nM Ac-WEHD-CHO (lane 3), CPVAcrmA::lacZ extracts
preincubated with 10 nM Ac-DEVD-CHO (lane 4), CPVAcrmA::lacZ extracts
preincubated with 200 pM Z-VAD-FMK (lane 5), CPVAcrmA::lacZ extracts alone (lane
6), CPVAcrmA::lacZ extracts preincubated with 1U of human caspase-1 (lane 7),
CPVAcrmA::lacZ extracts preincubated with 100 nM of Ac-WEHD-CHO and caspase-1
(lane 8), CPVAcrmA::lacZ extracts preincubated with 10 nM Ac-DEYD-CHO and
caspase-1 (lane 9) or CPVAcrmA::lacZ extracts preincubated with 200 pM Z-VAD-
FMK and caspase-1 (lane 10). The protein mixtures were resolved by electrophoresis on
10% SDS-PAGE and visualized by autoradiography. Radiolabeled peptides are: chicken
proIL-18 23 kDa; processed forms 19 kDa.


59
production of progeny particles. Virus infection itself generates proapoptotic signals.
Adsorption of non-infectious virus particles to the cell surface can trigger apoptotic
signaling. Avian leukosis virus enters cells via the TVB receptor, (a member of the DR
family with significant homology to human DR-5) which has been shown to transduce
apoptotic signal (48). Viral mechanisms that block both the extrinsic and intrinsic
pathways of apoptosis have been identified. The mitochondrial pathway can also be
triggered following virus infection. Viral replication cycle activates cell cycle regulators
such as p53 is thought to involve the Bcl-2 family of proteins to induce mitochondrial
dysfunction (131). Respiratory syncitial virus can activate the ER resident caspasel2
(37). The identification of viral inhibitors in both pathways of programmed cell death
indicates the importance of apoptosis in controlling a viral infection. Inhibitors of both
the intrinsic and extrinsic pathways are discussed.
Viral inhibitors of Mitochondria: PT pore modulators and Bcl-2 homologs
Recently vaccinia virus was shown to inhibit apoptotic induction mediated by
mitochondria (444). Infections of Jurkat cells with vaccinia virus deleted for caspase
inhibitor SPI-2 were found to be resistant to Fas-induced cell death, prevent the loss of
mitochondrial membrane potential and decrease cytochrome c levels as well as caspse-3
activity. The mechanism of vaccinia virus inhibition of mitochondrion mediated
apoptosis is not clearly understood but is believed to involve the ability of the virus to
modulate the permeability transition pore. The Hepatitis B virus HBx protein binds
human VDAC and inhibits apoptotic induction by altering transmembrane potential
(324). Viral mitochondria localized inhibitor of apoptosis (vMIA) encoded by


67
By far the most infamous poxvirus is variola, the causative agent of smallpox
infections in humans. Smallpox produced a systemic disease with a generalized rash in
humans and has been documented for about 2000 years (102). Of importance is that it is
the first and only human disease to date that has been purposefully eradicated. The
concept of variolation i.e. inoculation of dried smallpox scab material into the skin to
induce mild disease and subsequent immunity was practiced since the early 10th century
(102). It was not until 1798 when Edward Jenner demonstrated that inoculation with
cowpox could protect against smallpox. This new method, vaccination, quickly gained
widespread use and eventually led the eradication of smallpox in 1977 as a result of mass
vaccination programs carried out by the World Health Organization.
Poxvirus Lifecycle
Poxvirus genomes are linear double stranded DNA molecules that are covalently
joined at the termini. The internal core region of the genome is highly conserved among
poxviruses whereas variation occurs at the ends of the genome within the inverted
terminal repeat (ITR) regions. Genes located in this region are typically not essential for
viral growth in tissue culture, but contribute to viral pathogenesis in vivo. Poxviruses are
unique in that the entire life cycle is accomplished in the cytoplasm of the infected cell
(Figure 3). The receptor that mediates viral entry is unknown at present. However viral
entry is mediated by membrane fusion that is dependent on pH. Viral gene expression
occurs in three stages. The early genes are transcribed within the viral core that contains
all the transcription machinery. These include growth factors, immune defense
molecules such as CrmA and factors required for DNA replication. Intermediate genes


119
hours post infection (Fig. 10). As in the case of SPI-2, there were also present
cleaved/degraded forms of CrmA produced during RPV infections, although to a much
lesser extent.
CrmA expressed by wt CPV migrated as expected (Fig. 11). SPI-2 expressed
from CPV in place of CrmA was stable through out virus infection as the converse
construct seen in (Fig.l 1). SPI-2 expressed in CPV did not seem to be degraded/cleaved
as in wt RPV infections (compare Fig. 10 and 11). It is apparent that SPI-2 and CrmA
have been successfully swapped or interchanged between RPV and CPV, respectively,
and that the mutant viruses express the recombinant proteins.
The replacement of SPI-2 with CrmA in RPV and the converse construct in CPV
allowed us to examine the effect of the activity of these genes within different viral
contexts during infections of LLC-PK1 cells.
RPVASPI-2::crmA and CPVAcrmA::SPI-2 Prevent Apoptosis in Pig Cells
In order to test the equivalency of the SPI-2 and CrmA we swapped the genes
in the parent viruses and used the recombinant viruses to infect swine cells. Infected cells
were stained with DAPI and viewed microscopically as shown in Figures 12 and 13.
Consistent with previous reports, wt CPV-infected cells do not appear apoptotic whereas
CPVAcrmA::lacZ induced the morphological changes seen in apoptotic cells such as
densely staining apoptotic bodies (Fig. 13) (236). SPI-2 replacement of CrmA in CPV
behaved like wt CPV and did not induce apoptotic changes in swine cells suggesting that
SPI-2 could functionally substitute for CrmA function in CPV (Fig. 13). Both RPV and
RPVASPI-2 infections induced the formation of apoptotic cells in agreement with


98
emergence of the parental and recombinant virus. In addition to TDS, the use of a
screening marker such as P-galactosidase aids in identifying recombinant virus.
As described above, following transformation and plating of virus in the presence
of MPA, single viral plaques were selected during the first plaque pick. Subsequent
rounds of plaque purification were performed in the absence of MPA to allow for
concatemer resolution. At this stage of plaque purification, it would be useful to be able
to use P-galactosidase expression as an aid to select for plaques in the absence of MPA.
If the recombinant virus being constructed expresses P-galactosidase, then one would
screen for blue plaques in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-P-D-
galactopyranoside) (50 pi of 20 mg/ml X-Gal stock added to overlay of each well in 6-
well dish 12 hours prior to plaque picks). Alternatively, if the parental virus expressed P-
galactosidase, the recombinant virus was designed in such a way as to replace the P-
galactosidase marker and therefore one would select for non-P-galactosidase expressing
plaques or those that would be white instead of blue in the presence of X-Gal stain.
In the absence of any additional screening tool (such as p-galactosidase), multiple
MPA resistant plaques (typically 15) were picked in the first round of plaque purification.
Each plaque was purified independently. After 5 rounds of plaque purification, each
independently derived virus was grown in 60 mm dishes, and viral DNA was isolated.
The identification of recombinant virus was performed following PCR amplification of
the foreign gene from viral DNA. Typically the ratio of parental virus to recombinant
virus was consistently at approximately 9:1 respectively.


CHAPTER 2
MATERIALS AND METHODS
Virology
Cells
Primary chicken embryo fibroblasts (CEF) were obtained from 11 day old
embryonated chicken eggs according to standard methods. CEFs were maintained in
Medium 199 (Life Technologies, Grand Island, NY) supplemented with 2mM glutamine,
0.1 mM non-essential amino acids, ImM sodium pyruvate, 50U/ml penicillin, 50|ig/ml
streptomycin (Mediatech, Herndon, VA) and 5% fetal bovine serum (Life Technologies).
African Green monkey kidney (CV-1, ATCC CCL-70) cell line was maintained in
Minimum Essential Medium (MEM) with Earles salts (Life Technologies) and
supplements. Pig kidney (LLC-PK1, ATCC CL-101) cells were maintained in Medium
199 (Life Technologies) and supplements.
Viruses
Wild type cowpox virus (CPV strain Brighton Red; ATCC VR-302) and
rabbitpox virus (RPV strain Utrecht, ATCC VR-157) stocks were grown in CV-1 cells.
In this study, derivatives of CPV or RPV were made in which only the coding region for
CrmA/SPI-2 was replaced such that the regulatory elements were left intact.
87


inflammatory cytokines would be consistent with the role of CrmA in controlling
inflammation. CrmA also prevents apoptosis, consistent with the demonstrated inhibition
of caspases-8 and -10. The myxoma virus serpin, SERP2, has been shown to inhibit
caspases-1, -8 and -10 as does the non-serpin caspase inhibitor, P35, from baculovirus.
However, CPV recombinants where CrmA is replaced with SERP2, P35 or CrmA D303A
(mutation of the reactive center loop (RCL) which alters substrate specificity) all produce
white, inflammatory pocks on CAMs. Virus yields are restored from the P35 and SERP2
but not CrmA D303A recombinants. Pro-apoptotic caspase-3-like activity is detected
only from CPVAcrmA::lacZ and CPVCrmA D303A infections suggesting that loss of
functional CrmA leads to apoptosis and it is apoptosis, not inflammation which is
responsible for the reduction in virus yield. We did not detect caspase-1 within infected
CAMs through processing of chicken proIL-P or proIL-18, suggesting that caspase-1
mediated control of inflammation is unlikely. We show for the first time that the
regulation of apoptosis and inflammation are two distinct functions of CrmA that may not
depend solely on caspase inhibition.
xvi


24
induce an innate response as well as stimulate the production of a number of
inflammatory cytokines underscores its importance as a proinflammatory mediator.
Both IL-18 and IL-18R have been targeted for gene disruptions. IL-18 deficient
mice have impaired IFNy production, defective NK cell activity and reduced ThI
response (399). The importance of IL-18 in controlling intracellular pathogens was
demonstrated in IL-18A mice infected with the protozoan parasite Leishmania that were
highly susceptible to infection compared to wild type controls (445). Infections with the
extracellular bacteria Stapylococcus aureus caused reduced septicemia whereas these
mice developed more severe septic arthritis, a reaction associated with impaired ThI
response (445). Receptor knockout mice have also been characterized. IL-18_/' cells
from these mice had defects in NK cytolytic activity, IFNy production and were impaired
in NF-kB or JNK activity (157). The IL-18R'/_ mice also failed to respond to IL-18
indicating the essential role of this molecule in mediating signaling.
The availability of mature IL-18 under diverse conditions can determine the
outcome of the immune response. While IL-18 is processed to maturity by caspase-1 at
Asp36, there also exist alternative processing mechanisms. In addition to caspase-1,
caspases-4 and -5 have also been shown to process proIL-18 correctly (122). Caspase-3
on the other hand processed IL-18 at Asp71 and Asp76 resulting in polypeptides with no
significant biological activity (7, 133). Proteinase-3 (PR-3), a serine protease present in
neutrophil granules has also been shown to process proIL-18 (99, 389). Both membrane
bound and soluble forms of PR-3 enzyme have been identified. PR-3 has been associated
with a number of inflammatory diseases and has recently been implicated as one the
major autoantigens in Hepatitis C virus infection (47, 99, 348, 453). Interestingly PR-3


185
caspase-1 in vitro (331). Caspase-1 processes proIL-ip and proIL-18 to the mature pro-
inflammatory cytokines (85). CrmA as mentioned earlier can also inhibit the induction of
apoptosis by ectopic expression (93, 117, 407) and during CPV infection of pig kidney
cells (236, 332). These observations correlate with studies showing that CrmA inhibits
the apical caspases -8, -9 and -10 (118). Thus CrmA regulates both inflammation and
apoptosis during CPV infection. Results from our study are summarized in Table 7.
Requirement of Intact RCL for CrmA Function In Vivo
In order to determine if there are other regions within CrmA that may contribute
to its function during CPV infection, we mutated the PI Asp residue in CrmA RCL to
Ala. Mutation at the PI position of a serpin alters target specificity (368). Infections of
CAMs with CPVCrmA D303A recombinant virus produced white, inflammatory lesions
(Fig. 21). The CrmA D303A mutant pocks had essentially the same characteristics as
those from the null mutant infections with respect to inflammatory cell influx (Fig. 22),
virus yields (Fig. 24), caspase induction (Fig. 26) and the ability of pock extracts to
process proIL-ip and proIL-18 (Figs. 27-29). Therefore, we have shown for the first
time that CrmA functions by a protease inhibition mechanism in vivo and that serpin
function is required in order to inhibit inflammation and apoptosis on CAMs.
Regulation of Inflammation and Apoptosis by CrmA are Distinct Functions
We replaced the CrmA gene within CPV with other caspase inhibitors to
determine if CrmA functions solely by inhibiting caspases. If the preceding hypothesis
were true, P35 and SERP2 replacements of CrmA in CPV should behave like wild type


91
DNA Manipulation, Ligation and Transformation
Plasmid or PCR amplified DNA was digested with restriction endonucleases
according to manufacturers instructions (New England Biolabs). Digested DNA (1 pg
of plasmid DNA or 100 pi of PCR product) was purified after agarose gel electrophoresis
using the Geneclean II kit (Bio 101, Vista, CA) according to manufacturers instructions.
The purified DNA was quantified in a TD-700 fluorimeter (Turner Designs, Sunnyvale,
CA). Vector and insert DNA mixed at 1:2 and 1:10 ratios, respectively, were ligated in
20 pi volumes using 50 to 100 ng total DNA and T4 DNA ligase (New England Biolabs)
according to manufacturers instructions. Ligations were incubated at 16C overnight.
The entire ligation mixture was transformed into electrocompetant E.coli DH5a cells as
described by Sambrook (351). Transformed colonies of E.coli cells were grown on LB
agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% Difco Bacto agar; Becton
Dickinson, Sparks, MD) containing the appropriate antibiotic for selection. Transformed
colonies were screened by colony PCR using the cells directly as templates.
Briefly, using sterile toothpicks, single colonies were picked and streaked onto
LB agar plates (containing the appropriate antibiotic for selection), the toothpick was
then immersed in PCR reaction mixture (containing PCR buffer, primers specific for the
cloned gene and enzyme) that had been previously aliquoted into PCR tubes. The PCR
reaction was then carried out as described earlier. An aliquot of the PCR product
(typically 10 pi) was electrophoresied on 1% agarose gels. The presence of the cloned
gene in transformed colonies was confirmed by visualizing the PCR product under UV
light. Alternatively transformed colonies were screened by restriction enzyme digestion


36
MC54L bound both human and murine IL-18 with high affinity and prevented IL-18
mediated IFNy induction in KG-1 monocytic cells. IL-18 binding activity was found in
culture supernatants of a number of vaccinia strains as well as cowpox and ectromelia
viruses (377). Furthermore vIL-18BP of ectromelia virus was found to inhibit NF-kB
activation and IFNy induction in KG-1 monocytic cells in response to IL-18. Cowpox
and ectromelia viruses are rodent pathogens and not surprisingly their vIL-18BPs
preferentially bound mouse IL-18 over human IL-18 (52). Mice infected peritonealy
with ectromelia deleted for vIL-18BP had increased NK cell cytotoxic activity while the
mutant virus was found to be attenuated (43). IL-18 activity clearly is an important host
response mechanism seeing that a number of viruses block its activity.
Chemokine Inhibitors
The number of mechanisms employed by different viruses to subvert chemokine
activity indicates the importance of this response to control viral infection. Similar to
counter strategies used against cytokines, different herpes and poxviruses encode
molecules that regulate chemokines. Typically these fall into three categories. First are
molecules that are ligands or viral homologs of cellular chemokines. These include
human herpes virus proteins such as vMIP-I, vMIP-II, vMIP-III, that are homologs to
MIP-I a or p and function as agonists or antagonists (213, 264, 385). Cytomegalovirus
proteins vCXC-1 and -2 as well as Mareks disease virus vIL-8 are homologs of IL-8
(307, 312). Interestingly vIL-8 was found to chemoattract blood mononuclear cells but
not heterophils. Molluscum encodes two proteins that act as antagonists to MCP-1. The


115
Plasmid Containing Mouse ProIL-ip
A cDNA clone of mouse proIL-ip was kindly provided by Rudy Beyaert,
University of Ghent, Belgium. The orientation of the construct pGEMl 1-ILlbeta
containing mouse proIL-ip was such that transcription was possible using the SP6
promoter within the plasmid. In vitro radiolabeled proteins were synthesized using the
SP6 TNT system (Promega) according to manufacturers instructions.
Quick Coupled Transcription/Translation System
The Quick Coupled Transcription/Translation System (TNT) (Promega) is a
single-tube reaction for in vitro transcription and translation from plasmid constructs
containing the appropriate promoters for RNA polymerases such as SP6 and T7. The
TNT system contains a single mixture of RNA polymerase, nucleotides, salts, RNAse
inhibitors and rabbit reticulocyte lysate to form a master mix. Briefly, the reactions were
performed as follows. Typically 0.5-1 pg of plasmid DNA was added to 20 pi of
aliquoted TNT master mix. To this mixture was added [35S] methionine (10 pCi) (ICN),
and the volume brought up to 25 pi. The TNT reaction was incubated at 30C for 60
minutes. After the reaction is complete, the tubes were placed on ice if used immediately
or frozen at -20C until used. Typically for constructs in pTMl background, 1 to 2 pi of
TNT reaction was sufficient to be visualized following SDS-PAGE and autoradiography.
In Vitro Cleavage Assay for Processing ProIL-ip and ProIL-18
Cytokine processing activity was determined as follows. Two microliter of 35S-
labelled proIL-ip or proIL-18 from 25 pi TNT reaction was incubated with 1 unit of


159
Chicken ProIL-ip Processing Activity in CAMs.
Caspase-1 is responsible for the maturation of IL-ip and IL-18 (both are
inflammatory cytokines) from their respective precursors. The only infected CAM
extracts in which we observed any caspase activity, was in either CPVAcrmA::lacZ or
CPVCrmA D303A. Within those extracts, all the caspase activity we observed within
infected CAMs (Fig. 26 Insert) resembled caspase-3, consistent with the inability of these
viruses to control apoptosis. We have not been able to detect caspase-1-like activity
within infected CAMs using peptide substrates. However, a more sensitive assay for
caspase-1-like activity is cleavage of radiolabeled proIL-ip or proIL-18 precursor
molecules to active cytokines. Like CrmA, SERP2 and P35 also inhibit caspase-1.
Caspase-1 is believed to be involved in the processing of proIL-ip or proIL-18 cytokine
precursors through cleavage of the pro-inflammatory cytokine precursors at conserved
aspartate residues (99). The fact that CPVAcrmA::SERP2 and CPVAcrmA::P35 viruses
fail to control inflammation casts some doubts that inflammation is caspase mediated.
Examination of the avian and mammalian gene sequences raised further questions
in this regard. The activation of mammalian proIL-ip is characterized by caspase-1
mediated cleavage at Asp27 followed by cleavage at Asp 116, sites generally conserved
in mammals including humans (395). However, inspection of the chicken proIL-ip gene
showed both these critical aspartic acids to be missing (446). Although there are
neighboring aspartic acids present, they are not within the conserved caspase-1 cleavage
site context.
A cDNA clone of chicken IL-1 (3 which encoded amino acids 106 to the C-
terminus, was found to be active (446). The molecule was designed based on the


I am grateful to Joyce Conners for taking care of all the necessary paperwork. I
would also like to thank the administrative staff of the Department of Molecular Genetics
and Microbiology for their timely help.
I owe a lot to friends at the Graduate Evangelical Fellowship, who have seen me
through my graduate career. I would specially like to mention Chip Appel, whose
constant encouragement and friendship was invaluable at times. Brian and Shigeko
Jackson, Diomy Zamora, Kwabena Amphosa-Managera, Shelly Merves and Ricardo
Gomez have specially seen me through the final stages of completing this project. Their
friendship has been invaluable. My family, have patiently waited for me to finish this
degree and have been a positive influence through all these years. Finally, I would like to
express my heartfelt gratitude to my wife, Leena, for her love, patience and endurance
despite all the strain we have gone through my graduate career. Leena has truly been my
helpmate. I firmly believe in the notion that it takes a village to raise an individual.
There are many others, who have, in little ways influenced my life and shaped the way I
think. Like little drops of water that make a flowing river, these individuals have touched
my life in numerous ways.
m


14
and chronic states of inflammation. In summary, a number of proteases other than
caspase-1 can cleave pro-IL-ip to generate the active cytokine.
A number of knockout mice models in the IL-1 system has been studied and are
listed in Tables 1 & 2. IL-la is the least well studied, and observations indicate that it is
not involved in the inflammatory responses induced by turpentine or infection with
Listeria (98, 155). IL-1 p is crucial for the development of an acute phase response and
in the induction of febrile response to inflammation (472). Quite unexpectedly IL-1 P/_
mice were hypersensitive to both IL-a and IL-1 P, an observation that so far has not been
explained (12). Mice deleted for IL-Ra were seen to spontaneously develop autoimmune
disorders that resembled rheumatoid arthritis, arterial inflammation and were shown to be
highly susceptible to LPS induced lethality (149, 156, 290). IL-1RI knockout mice failed
to induce a biological response to either IL-la or p, indicating that this receptor is
essential for all IL-1 activity (126, 212). In IL-lRAcP7' fibroblasts, binding of IL-la or
IL-IRa was only moderately reduced, whereas IL-ip binding affinity was reduced 70-
fold indicating the requirement of accessory protein for efficient receptor binding (77).
Similarly these mice failed to induce a biological response to IL-1. Disruption of the
signaling component IRAK caused reduced activation of NF-kB in fibroblasts and
attenuated responses to IL-1 in mice (410). Caspase-17' mice are deficient in processing
IL-ip (209). Other studies have shown mature IL-1 p to be released despite caspase-1
deficiency (100). Although caspase-1 is the most important protease required for IL-ip
maturation other enzymes have been implicated to perform this function. The importance
of enzymes other than caspase-1 in IL-1 p maturation may be important in certain cases
and will be discussed in relation to the results presented in this dissertation. These


16
Table 2. List of mice deficient in the IL-1 system.
IL-1 system component
Mouse strain (reference)
IL-1RI
C57BL/6 x 129J (126); C57BL/6 x 129/SV (212)
IL-lRAcP
C57BL/6 (77)
IL-1 a
C57BL/6x DBA/2 (155)
IL-13
C57BL/6 x DBA/2 (155); C57BL/6 x 129EV (100)
IL-1 receptor antagonist
C57BL/6 (149, 290); MF-1 (290)
IRAK
C57BL/6 (410)
Caspase-1
C57BL/6x 129(100)


This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
May 2003


BIOGRAPHICAL SKETCH
Rajkumar Nathaniel was bom on April 13, 1971 in Madras, India. He and his
brother Vijaykumar grew up in Mysore, India. He completed the Bachelor of Science
degree from St. Josephs College at Bangalore in 1991. After his graduation he was
trained as a Medical Microbiologist at the Christian Medical College, Vellore, India.
From 1993 to 1994 he worked as a Research Assistant at AstraZeneca Research,
Bangalore. In the Fall of 1994, he joined the graduate program at the Department of
Microbiology, University of Central Florida, Orlando, where he obtained the Master of
Science degree. In 1996 he joined the Interdisciplinary Program in Biomedical Sciences
at the University of Florida. He entered Dr. Richard Moyers laboratory in the
Department of Molecular Genetics and Microbiology in the Summer of 1997 to begin
work on his dissertation. He married Mamatha Chandrakumar in 1999. Following the
completion of his Ph.D., he joined the laboratory of Dr. Richard Benya at the University
of Illinois at Chicago to begin postdoctoral training.
239


228
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ii
LIST OF TABLES viii
LIST OF FIGURES ix
ABBREVIATIONS xi
ABSTRACT xv
CHAPTER
1 LITERATURE REVIEW 1
Introduction 1
Immune Systems 1
Innate Immunity and Inflammation 3
Innate Immune Recognition 5
Toll and Interleukin-1 Receptors 6
Toll and Toll Like Receptors (TLR) 6
Interleukin-1 (IL-1) 8
Inflammatory Mediators 17
Tumor Necrosis Factor (TNF) 17
Interleukin-18 (IL-18) 21
Interleukin-8 (IL-8) 25
Avian Cytokines 29
TNF-a 29
IL-1 30
IL-18 32
CXC Chemokines 32
Viral Inhibitors of Inflammation 33
TNF Inhibitors 33
IL-1 Inhibitors 34
IL-18 Inhibitors 35
Chemokine Inhibitors 36
Inhibitors of Complement 38
Inhibitors of Interferons 39
Apoptosis 40
IV




101
polymerase using primers FS 129 (5-
GTCAGATCCATGGTTGAATTCCGAGCTTGGCTG-3 ) containing the Ncol site and
FS 130 (5-AGTCAACGCCCGGGTACGCTCACAGAATTCCCG-3) containing the
Smal site. The lacZ PCR product was cloned into the Ncol and Smal sites of pBS-
RgS/A resulting in pRglacZ. Using pRglacZ the SPI-2 open reading frame in CPV was
replaced with lacZ by transient dominant selection (97) to make RPVASPI-2::lacZ.
A clone of the P35 gene from the baculovirus Autographa californica NPV was
provided by Lois Miller (University of Georgia, Athens). The plasmid pR35 was
constructed by inserting the P35 coding region between the Ncol and Smal sites of pBS-
RgS/A by recombinant PCR (421) such that the start codon of P35 exactly replaces the
ATG at the Ncol site. The primer pair used to generate the left flank was FS 1 (5-
GATCTCT AGAGCGGCCGCGGTTCGGT GGC A AACTTAC AT GGA A-3 ) containing
an Xbal site (underlined) and FS 90 (5-AGTAATCGATATTGGTCGTG-3 ). The
primer pair used to generate P35 was FS 88 (5-
CACGACCAATATCGATTACTATGTGTGTAATTTTTCCGG-3 ) (underlined portion
is complimentary to FS 90) and FS 74
(5 T AC GT C ACCCGGGTT ATTT AATT GTGTTT AAT ATTAC-3) containing a Smal
site at the 3end (underlined). Finally the left flank was linked to P35 using primers FS 1
and FS 74. The full length PCR product was cloned into the Xbal and Smal sites of pBS-
RgS/A resulting in pR35. The P35 recombinant virus RPVASPI-2::P35 was made using
pR35 and RPVASPI-2::lacZ by transient dominant selection and X-Gal staining of
recombinant viral plaques.


187
To address the issue of whether the inflammation seen on the CAMs was indeed
dependent on and mediated by caspase-1, we assayed extracts for the ability to process
the two capase-1 substrates proIL-18 and proIL-1P (Figs. 27, 28 and 29). Caspase-1, like
all other caspases specifically cleaves substrates after certain Asp residues. Unlike the
mammalian system where maturation of IL-ip requires caspase-1 to process the inactive
precursor initially at Asp27 (site 1) followed by cleavage at Aspl 16 (site 2) (395), the
chicken proIL-ip sequence does not contain either Asp27 or Aspl 16 residue required for
cleavage by caspase-1 that would release the mature cytokine (446). The mechanism of
IL-1P maturation in chickens is currently unknown. The chicken proIL-1 P sequence is
similar to proIL-ip sequences from frog and fish, which also do not contain the
conserved Asp sites found in mammals (475). Biological activity has been shown with a
putative chicken IL-ip recombinant protein (446). While chicken caspase-1 shares 44%
homology to mammalian caspase-1 (178), there is no functional data to show processing
of either chicken proIL-18 or proIL-1 P by chicken caspase-1.
Indeed, the failure of recombinant human caspase-1 to process chicken proIL-ip
(Fig. 27) is also consistent with the idea that the mechanism of avian IL-1 p activation
may not be mediated by caspase-1. If the mechanism of processing chicken proIL-ip
involved an alternate non-caspase-1, then one would expect the inflammatory extracts of
CPVAcrmAdacZ, CPVCrmA D303A, CPVAcrmA::P35 and CPVAcrmA::SERP2 to
process the chicken proIL-1 p precursor. We found no evidence for chicken proIL-1 p
processing from any infected extract (Fig. 27). Since we were assaying for chicken
caspase-1 activity, we chose to use an alternate caspase-1 substrate namely mouse proIL-
1P which contains the conserved Asp residues required for processing by caspase-1. We


38
primary infection (215). More recently M-Tl was found to differ from rabbitpoxvirus 35
kDa protein in that M-Tl can localize to cell surfaces through interactions at the C-
terminus of the molecule and heparin (363). The breadth of viral modulators of
chemokines suggests that chemotaxis is an important host response to virus infection.
Inhibitors of Complement
Activation of complement pathways can itself lead to the destruction of virus-
infected cells as well as mediate the release potent inflammatory cytokines and is another
mechanism targeted by virus infections. Mammalian regulators of complement
activation function to protect bystander cells from the effects of activated complement.
These regulators are characterized by homologous motifs termed short consensus repeats
(SCRs) (335). The vaccinia complement control protein (VCP) contains four SCRs and
regulates complement by binding to C4b and C3b (204, 251). VCP acts as a cofactor to
the cellular complement regulator serine protease factor I and aids in the cleavage of C4b
and C3b to inactive components (349). The cowpox VCP homolog called
immunomodulatory protein (IMP) was also shown to be functional in inhibiting tissue
damage due to complement at the site of viral infection both in the footpad and
subcutaneous models of mice injections (158, 258). More recently the variola homolog
of VCP termed smallpox inhibitor of complement systems (SPICE) was found to be
nearly 100 fold and 6 fold more potent at inactivating human C3b and C4b respectively
than its vaccinia counterpart (341). Glycoproteins C (gC) of human herpes virus 1 and 2
have also been found to bind C3 components of complement (203). In addition the
herpesvirus saimir CCPH gene product prevents complement mediated cell damage and


203
78. Damon, I., P. M. Murphy, and B. Moss. 1998. Broad spectrum chemokine
antagonistic activity of a human poxvirus chemokine homolog. Proc. Natl. Acad.
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80. Deveraux, Q. L., N. Roy, H. R. Stennicke, T. Van Arsdale, Q. Zhou, S. M.
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82. Dinarello, C. A. 1996. Biologic basis for interleukin-1 in disease. Blood 87:2095-
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83. Dinarello, C. A. 1997. Interleukin-1. Cytokine Growth Factor Rev. 8:253-265.
84. Dinarello, C. A. 1998. Interleukin-1, interleukin-1 receptors and interleukin-1
receptor antagonist. Int. Rev. Immunol. 16:457-499.
85. Dinarello, C. A. 2000. Interleukin-1 beta and interleukin-18: Two cytokine
precursors for interleukin-1 beta converting enzyme (Caspase-1). Immune
Response in the Critically Ill 31:84-96.
86. Dinarello, C. A. 2000. The role of the interleukin-1-receptor antagonist in
blocking inflammation mediated by interleukin-1. N. Engl. J. Med. 343:732-734.
87. Du, C., M. Fang, Y. Li, L. Li, and X. Wang. 2000. Smac, a mitochondrial
protein that promotes cytochrome c-dependent caspase activation by eliminating
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88. Duan, H. and V. M. Dixit. 1997. RAIDD is a new 'death' adaptor molecule.
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89. Duckett, C. S., F. Li, Y. Wang, K. J. Tomaselli, C. B. Thompson, and R. C.
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90. Earnshaw, W. C., L. M. Martins, and S. H. Kaufmann. 1999. Mammalian
caspases: Structure, activation, substrates, and functions during apoptosis. Annual
Review of Biochemistry 68:383-424.


106
The P7 5-gpt cassette which confers resistance to mycophenolic acid (96) was
derived from pBS-gptA (421) and PCR amplified amplified with Vent polymerase
(Amersham) using primers FS 414 (5-
TCAGACTGCAGCCCGGTAGTTGCGATATAC-3) containing the PstI site
(underlined) and FS 415 (5 -TGACGCCCGGG AG ATTAGCG ACCGGAG ATT-3 )
containing the Smal site (underlined). The Eco.gpt PCR product was cloned into the PstI
and Smal sites of pBS-CPVIL-lR-LF to generate pBS-CPVIL-lR-LF-MPA.
The right flanking sequence was PCR amplified from CPV genomic DNA using
primers FS 416 (5 -AGCTGCCCGGGCGTTGAGACCTCCCACAACG-3 ) containing
the Smal site (underlined) and FS 408 (5-
CTGC ACGGATCCC AATC ACGTTATACTAATAGTAAC-3 ) containing the BamHI
site (underlined). The right flanking PCR product was cloned into the Smal and BamHI
sites of pBS-CPVIL-lR-LF-MPA resulting in pBS-CPVIL-IRKO.
By mycophenolic acid selection, CPV AIL-1 pR::Eco.gpt virus was created using pBS-
CPVIL-1RKO and wt CPV.
Apoptosis Assays
DAPI Staining of Infected Cells
Cells undergoing apoptosis were visualized by staining DNA within cells. LLC-
PK1 cells were grown to 80% confluency in eight-well chamber slides (LabTek,
Campbell CA) and infected at an MOI of 10. After 2 hour adsorption, the inoculum was
removed, and 200 pi of Medium 199 was added. The cells were further incubated for 14
hours and then washed with 300 pi of PBS. Cells were fixed in 200 pi of PBS containing


168
Chicken
Pro-IL-18
Processed
1 2 3456789
Figure 30. Caspase-1 mediated processing of chicken proIL-18 is blocked by
peptides. 35S labeled chicken proIL-18 proteins synthesized in vitro were incubated with
either buffer (lane 1), 1U of human caspase-1 (lane 2), caspase-1 preincubated with 100
nM Ac-WEHD-CHO (lane 3), caspase-1 preincubated with 10 nM Ac-DEVD-CHO
(lane 4), caspase-1 preincubated with 200 pM Z-VAD-FMK (lane 5), 15U of human
caspase-3 (lane 6), caspase-3 preincubated with 100 nM Ac-WEHD-CHO (lane 7),
caspase-3 preincubated with lOnM Ac-DEVD-CHO (lane 8) or caspase-3 preincubated
with 200 pM Z-VAD-FMK (lane 9). The protein mixtures were resolved by
electrophoresis on 10% SDS-PAGE and visualized by autoradiography. Radiolabeled
peptides are: chicken proIL-18 23 kDa; processed form 19 kDa


158
caspase-3, -7 and -8 and Z-VAD-FMK is an inhibitor of all caspases (118). The
minimum concentrations of Ac-WEHD-CHO and Ac-DEVD-CHO determined to give
complete inhibition of human recombinant caspase-1 (1U) and caspase-3 (10U) was
determined to be lOOnM and lOnM respectively based on hydrolysis of fluorogenic
substrates (data not shown).
Extracts were made from confluently infected CAMS and pre-incubated for 2 hrs
in the presence of increasing concentrations of either Ac-WEHD-CHO or Ac-DEVD-
CHO and thereafter assayed for caspase 3-like activity as in Figure 26 using the
fluorogenic substrate Ac-DEVD-AMC (Fig. 26 Insert). The caspase-1 inhibitor, Ac-
WEHD-CHO up to lOOnM has no effect on the cleaving activity present in the extracts
(data not shown), which was not surprising since the substrate used in these reactions,
Ac-DEVD-AMC does not measure caspase-1-like activity. However, the caspase-3-like
inhibitory peptide, Ac-DEVD-CHO gave marked inhibition of the activity as expected.
No cleavage activity in these extracts was observed when the caspase-1-like substrate,
Ac-WEHD-AMC is used in comparable assays, indicating an absence of detectable
levels of caspase-1-like activity (data not shown). Therefore, based on these assays, all
caspase activity observed in extracts of CPVAcrmA::lacZ infected CAMs, are solely due
to caspase-3-like activity.
P35 and SERP2 are able to function like CrmA to inhibit the induction of
apoptosis and restore virus yields within CAMs; however, unlike CrmA, fail to inhibit
inflammation. Therefore the replacement of CrmA by P35 and SERP2 can only partially
complement CrmA function. Thus, the regulation of inflammation and apoptosis by
CrmA are two distinct properties, and may not depend on caspase inhibition alone.


73
Figure 5. Serpin RCL. Position of the C-terminal reactive center loop (RCL) in the
serpin molecule. PI residue within RCL determines specificity for proteinase.


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Richard W. Moyer, C
Professor of Molecul
Microbiology
enetics and
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Molecular Genetics and
Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Lung-Ji Chang
Associate Professor of Molecular
Genetics and Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
WMl
Michael'T/Clare-Saflzi'er
Associate ProfessorVDPathology,
Immunology, and Laboratory
Medicine


229
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374. Slee, E. A., M. T. Harte, R. M. Kluck, B. B. Wolf, C. A. Casiano, D. D.
Newmeyer, H. G. Wang, J. C. Reed, D. W. Nicholson, E. S. Alnemri, D. R.
Green, and S. J. Martin. 1999. Ordering the cytochrome c-initiated caspase
cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-
9-dependent manner. J. Cell Biol. 144:281-292.
375. Smith, C. A., F. Q. Hu, T. D. Smith, C. L. Richards, P. Smolak, R. G.
Goodwin, and D. J. Pickup. 1996. Cowpox virus genome encodes a second
soluble homologue of cellular TNF receptors, distinct from CrmB, that binds TNF
but not LT alpha. Virology 223:132-147.
376. Smith, G. L. and Y. S. Chan. 1991. Two vaccinia virus proteins structurally
related to the interleukin-1 receptor and the immunoglobulin superfamily. J. Gen.
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377. Smith, V. P., N. A. Bryant, and A. Alcami. 2000. Ectromelia, vaccinia and
cowpox viruses encode secreted interleukin-18-binding proteins. J. Gen. Virol. 81
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378. Snipas, S. J., H. R. Stennicke, S. Riedl, J. Potempa, J. Travis, A. J. Barrett,
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379. Solomon, K. A., N. Pesti, G. Wu, and R. C. Newton. 1999. Cutting edge: a
dominant negative form of TNF-alpha converting enzyme inhibits proTNF and
TNFRII secretion. J. Immunol. 163:4105-4108.


Ill
sac. Due to negative pressure created by the deflated air sac, the chorioallantoic
membrane detached from the shell and was suspended below the second incision. Eggs
with dropped CAMs were always oriented with the CAM forming the apical surface.
Following aseptic introduction of virus onto the CAM, the incisions were sealed with
adhesive tape.
Infecting and Harvesting CAMs
The dropped CAMs were inoculated aseptically with 10 to 100 pock-forming
units (PFU) of virus in a lcc syringe tipped with 27.5 G needle. The inoculated eggs were
further incubated at 38.5C for 72 hours on a tabletop incubator without shaking. At the
end of the incubation period, the CAMs were harvested from the eggs. Briefly, the egg
was held over a disposal container, and the region of shell directly on the opposite side of
the CAM (underside of the positioned egg) was cracked open gently. Using a large
scissor, the shell was cut open slowly as the contents were allowed to collect in the
disposal container. The chick embryo was destroyed in the process to prevent the
embryo from pulling apart the entire CAM. The CAM was carefully detached from the
empty eggshell with a pair of forceps and placed in a petri dish containing phosphate
buffered saline (PBS) (pH 7.2). The harvested CAMs were washed twice in PBS and
scanned on a Microtek Scanmakerlll flatbed scanner at 600dpi. Individual pocks were
excised and stored at -80C. Alternatively, membranes inoculated with 500 PFU were
harvested at 48 hours post infection to yield confluently infected membranes, which were
stored at -80C till processed for extracts.


VAR
VV
TNFR
TRADD
variola virus
vaccinia virus
tumor necrosis factor receptor
TNFR associated death domain
xiv


Table 7. Summary of results using recombinant CPVs.
CPV
CPVAcrmA
::lacZ
CPVAcrmA
::P35
CPVAcrmA
::SERP2
CPVCrmA
D303A
CPV AIL-1 PR
"Eco.gpt
LLCPK1 Infection
DAPI
No apoptosis
Apoptosis
No apoptosis
Apoptosis
Apoptosis
ND
Terminal caspase
-
+
-
+
+
ND
CAM Infections
Pock morphology
Red
White
White
White
White
Red
Heterophil influx
-
+
+
+
+
ND
MTT/NBT reduction
-
+
+
+
+
-
Virus Titers
106
o
1
o
106
106
104-105
ND
Terminal caspase
-
+
-
-
+
ND
chIL-18 processing
-
+
-
-
+
ND
chIL-l(5 processing
-
-
-
-
-
ND
hCaspase-1 inhibition
Inhibits
No inhibition
Inhibits
Inhibits
No inhibition
ND
ND-not determined


164
When exogenous human caspase-1 was added to extracts from CPVAcrmA::lacZ and
CPVCrmA D303A infected CAMs, cleavage and maturation of mouse IL-ip was noted
(Fig. 28, lanes 3 and 6). However, when caspase-1 was added to extracts from CPV,
CPVAcrmA::P35 and CPVAcrmA::SERP2 infections (Fig. 28, lanes 2, 4 and 5), no
processing of mouse proIL-1p to mature IL-ip was noted implying that there remains
sufficient amounts of functional P35 and SERP2 respectively in those extracts to inhibit
the added caspase-1. Therefore, P35 and SERP2 would function like CrmA to prevent
caspase-1 activity were caspase-1 present adding further evidence to the theory that
caspase-1 mediated IL-1 p activation may not be the cause of inflammation on CAMs
during CPV infection.
Processing of Chicken ProIL-18 can be Blocked by either SERP2 or P35 within
Inflammatory Pocks
Unlike proIL-ip, chicken proIL-18 maintains the mammalian proIL-18 cleavage
site. Therefore we also tested the ability of CAM extracts to cleave radiolabeled chicken
proIL-18 (Fig. 29). Lysates were prepared from infected membranes and incubated with
35S radiolabeled chicken proIL-18. Processing activity was measured by the cleavage of
the precursor molecules monitored following separation of the products by SDS-PAGE
and subsequent autoradiography. Radiolabeled, control chicken proIL-18 migrated at
approximately 23 kDa (Fig. 29, lane 1). When incubated with 1U of recombinant human
caspase-1, the precursor proIL-18 peptide was processed to the mature cytokine, which
migrated at 19 kDa on SDS-PAGE (Fig. 29, lane 2) indicating that the cleavage site is


25
can also mediate the induction of apoptosis in HL-60 cells that is independent of caspase
activity and loss of mitochondrial membrane potential in spite of being implicated in the
cleavage and activation of caspase-3 (453). Of interest will be future studies on
understanding the biological activities of PR-3 since it seems to play a role in both
inflammation and apoptosis.
In summary, IL-18 induces the production of inflammatory cytokines (TNF-a,
IL-ip and IFNy), growth factors (GM-CSF) and chemokines (IL-8, MCP-1, MIP-la and
MIP-ip). The biological activities associated with IL-18 drive a ThI response, enhance
NK cell cytotoxicity and induce macrophage activation. Thus IL-18 plays a crucial role
in the innate immune response against pathogens such as viruses. The fact that
poxviruses such as cowpoxvirus encode molecules to bind IL-18; thereby disrupting
cytokine function, indicate the importance of IL-18 as an antiviral agent and mediator of
inflammation.
Interleukin-8 (IL-8)
IL-8 was originally identified as a neutrophil chemo attractant from supernatants
of activated monocytes (359, 437, 467). IL-8 is a 7 tolO kDa protein that belongs to the
CXC family of chemotactic cytokines termed chemokines and is now called CXCL8.
Chemokines are classified based on the position of the first two N-terminal cysteines that
can be separated by one amino acid (CXC) or can be adjacent (CC) (22). Disulphide
bonds between the first and third Cys as well as the second and fourth Cys are important
for three dimensional folding, receptor recognition and biological activity. IL-8 is
synthesized as a 99 amino acid precursor that is proteolytically processed by the removal


44
apoptosis. Finally, neighboring cells and/or macrophages circumvent an inflammatory
response by phagocytosing the vesicles or apoptotic bodies formed as a result of
apoptotic induction.
Caspases
Caspases are defined as cysteine proteases that cleave protein substrates after
specific aspartic acid or the PI residue (15). There are more than 42 mammalian
caspases described to date (217). Of these 12 are found in humans, caspases-1 to -10
(119), caspase-12 (105) and caspase-14 (91). There are also caspases cloned from
diverse organisms such as worms (459, 460, 468), insects (110, 168, 365, 380) and hydra
(66).
Caspases are thiol proteases in that they make use of a cysteine side chain
nucleophile to hydrolyze cleavage of the peptide bond. Like other proteases they are
produced as zymogens and maturation involves cleavage of the propeptide after certain
aspartate residues. Caspase precursors contain an N-terminal prodomain that can vary in
length from 23 to 219 amino acids followed by regions encoding a large (p20) and a
small (plO) subunit. Sometimes spacer regions can also be present between subunits that
are excised during the maturation process (90).
The association of one large and one small subunit (p20/pl0 heterodimer) gives
rise to the catalytic domain. Each active enzyme is a tetramer composed of two such
heterodimers. The quaternary structure of caspases is unique among proteases and is
described as a homodimer of heterodimers containing two active sites at opposite ends.
While the requirement for an aspartic acid residue at the PI position of the substrate is an


85
CrmA in CPV. Given its properties and the fact that SERP2 also has a PI Asp in the RCL
like CrmA, one would expect this myxoma protein to be the ideal poxvirus serpin
candidate to replace CrmA in CPV thereby addressing the third question. If SERP2 is
able to fully function like CrmA in CPV, this would indicate that serpin homologs having
identical PI residues from different poxviruses (at least in this case) have similar targets
in vivo and therefore perform similar functions.
Finally, we wanted to ascertain if IL-1 P plays a role in the inflammatory response
during CPV infections of the CAM. As mentioned earlier IL-ip can be produced by a
number of cell types in response to appropriate stimuli. Orthopoxviruses can control IL-
1P by two mechanisms. Generation of the mature cytokine can be blocked intracellularly
by CrmAs inhibition of caspase-1, while extracellular mature cytokine can be blocked
by the virus encoded IL-1P receptor. We deleted the viral IL-1P receptor in CPV and
used the resulting mutant virus (CPVAIL-ipR) to infect CAMs in order to determine
whether CPVAIL-1 PR infection inhibited or induced the formation of inflammatory
pocks. If IL-1 p were to be involved in CPV infections, one would expect the deletion of
the virus encoded IL-1 P receptor to induce an inflammatory response during CPVAIL-
1PR infections.
In this study, we show that despite their high degrees of homology SPI-2 and
CrmA are not functionally equivalent. The minute differences between the two proteins
can be recognized by different viral contexts and are amplified in the resulting infections.
We show for the first time that CrmA functions as a protease inhibitor to block
inflammation in vivo. We also show that P35 and SERP2 function like CrmA to inhibit
terminal caspase activity indicative of apoptosis during CPV infections of CAMs.


69
are transcribed concomitantly with DNA replication. Intermediate gene products are
factors for late gene transcription. Late gene products include structural proteins,
enzymes and early transcription factors. The viral DNA is then packaged within the
cytoplasmic viral factories and maturation proceeds to form infectious intracellular
mature virus (IMV) particles. IMV can be enveloped by membranes derived from the
golgi apparatus to form intracellular enveloped virus (IEV) which upon exiting the cell
looses a membrane layer and is termed extracellular enveloped virus (EEV).
Importance of Poxviruses
Poxviruses are extremely efficient in evading the effectors of antiviral immunity. Many
of these mechanisms target conserved cellular pathways. The knowledge gained from the
interactions of these viral proteins would be useful in understanding the immune response
to a poxvirus infection. Such studies also help in elucidating mechanisms of immune
evasion used by the virus. Therapeutic strategies based on models generated from viral
studies could be used to design molecules to modulate immune responses that occur in
other pathological conditions. Vaccinia virus is also an excellent vector for the
expression of foreign proteins, but the virus also elicits a tremendous immune response.
Therefore while the virus has potential as a vector in gene therapy, it might be
advantageous to minimize the side effects of treatment (immuneregulation) without
compromising gene delivery. Identifying and possibly modifying the genes responsible
for the vigorous immune response can improve its use in gene therapy. Finally, though
smallpox has officially been eradicated, interest in smallpox is sustained due to its
potential use as a weapon of mass destruction in biological warfare. Smallpox is also


162
Processing of ProIL-ip can be Blocked by either SERP2 or P35 within
Inflammatory Pocks
To ensure that our assay to detect caspase-1 activity was valid, we used an
alternative substrate such as mouse proIL-P in order to assay the chicken enzyme activity.
Extracts from infected CAMs were incubated with 35S radiolabeled mouse proIL-ip in
the presence or absence of recombinant human caspase-1. Processing activity was
measured by monitoring the cleavage of the precursor molecules with SDS-PAGE and
subsequent autoradiography. As seen in Figure 28, lane 1, native mouse proIL-lp
migrates as a peptide of 31 kDa. The mouse IL-1 p is processed as anticipated to an
intermediate 29 kDa peptide and then to the mature 17 kDa peptide by addition of human
caspase-1 (Fig. 28, lane 1). Again however, none of the infected CAM extracts were able
to process mouse proIL-ip (Fig. 28, lanes 2-6) to the mature form. We did, however, see
some partial processing of the mouse substrate with extracts from every infection except
wt CPV. Extended incubation (up to 6 hours) of the mouse proIL-1 P substrate with
CAM extracts did not yield mature IL-1 P from any infection condition (data not shown).
It is possible that the ability of CAM extracts to very minimally mediate partial
processing of mouse proIL-1 P but fail to completely maturate the mammalian cytokine
may be due to species specificity of the avian enzyme, i.e. a poor affinity of chicken
caspases for mouse proIL-ip. However, the question of species specificity could be
addressed using chicken proIL-18 as a substrate for chicken caspase-1 (see below).
Therefore within the limits of detectability, we found no evidence for the presence of any
caspase-1 activity in the infected CAMs against either mammalian or chicken proIL-ip.


167
DEVD-CHO (Fig. 30, lanes 1-5). Unlike previous reports (122, 133) we found little if
any indication for cleavage of proIL-18 by up to 15U of purified human caspase-3 to this
reaction (Fig. 30, lanes 6-9).
The inability of human caspase-3 to process chicken proIL-18 could be explained
due to species specificity, i.e. a low affinity of human caspase-3 for the chicken substrate.
However, we have also tested the ability of human caspase-3 to cleave mammalian
proIL-18 and found that up to 10U of recombinant human caspase-3 was unable to cleave
bovine proIL-18 (data not shown). Therefore the results in Figure 30 indicate that the
peptide inhibitors Ac-WEHD-CHO and Ac-DEVD-CHO were specific for caspases-1
and -3 respectively and that caspase-1 mediated cleavage of chicken proIL-18 is inhibited
by Ac-WEHD-CHO but not Ac-DEVD-CHO.
We then re-investigated the proIL-18 cleaving activity observed in the extracts
from CPVAcrmA::lacZ infected CAMs. Extracts were preincubated with the peptide
inhibitors prior to the addition of radiolabeled chicken proIL-18 (Fig. 31). Native
chicken proIL-18 migrates as a 23 kDa polypeptide (Fig. 31, lane 1). CPVAcrmA::lacZ
CAM extracts cleave chicken proIL-18 to the 19 kDa mature peptide (Fig. 31, lane 2).
Preincubation of extracts from CPVAcrmA::lacZ infections with Ac-DEVD-CHO or Z-
VAD-FMK but not Ac-WEHD-CHO blocked the processing of chicken proIL-18 (Fig.
31, compare lanes 2-5). Despite a failure of purified caspase-3 to cleave chicken proIL-
18 (Fig. 30), these results are most consistent with a terminal caspase mediated cleavage
of proIL-18. In addition to caspase-1 mediated processing of proIL-18, there also exist
alternative processing mechanisms to yield biologically active cytokine (122, 133, 389).
Therefore an alternative, a non-caspase enzyme inhibited by both Ac-DEVD-CHO and


135
analyzed P35 expression in the presence of cytosine arabinoside (AraC) (Fig. 17A).
AraC inhibits the synthesis of poxvirus DNA and thus prevents the expression of late
poxvirus proteins. Infected cell extracts prepared at early (6 hours) and late times (15
hours) post infection were immunoblotted for P35. As seen in Figure 17A, lanes 1 and 2,
P35 expressed in RPVASPI-2::P35 is present at 6 hours post infection both in the
presence and absence of AraC as expected although its expression in the presence of
AraC is increased. The increase in P35 can be explained by the ability of AraC to
prevent poxvirus DNA replication and thus late gene transcription leading to extended
early gene synthesis. At 15 hours post infection P35 was detected only in the presence of
AraC (Fig. 17A, lanes 3 and 4). However critical analysis of the data shows that the
relative decrease of P35 in the presence of AraC (Fig. 17A, lanes 1 and 4) is similar to
that in the absence of AraC (Fig. 17A, lanes 2 and 3), the only difference being higher
starting levels of P35 in the presence of AraC. This suggests that P35 is inherently
unstable within RPV infections and late events in viral replication may or may not be
responsible for decrease in P35 expression levels in RPVASPI-2::P35. Ac-DEVD-AMC
cleavage assays performed on extracts prepared from cells treated as mentioned reveal
that AraC blocks the induction of apoptosis (data not shown) indicating that apoptosis
occurs after/as a result of viral DNA replication.
At 6 hours post infection, P35 expressed in CPVAcrmA::P35 infections is seen
both in the presence and absence of AraC (Fig. 17B, lanes 1 and 2). However at 15 hours
post infection P35 expressed in the context of CPV is present even in the absence of
AraC (Fig. 17B, lanes 3 and 4) unlike in the case of RPVASPI-2::P35 infections (Fig.
17A, lanes 3 and 4). This clearly indicates that P35 is stable despite late events of virus


172
Figure 32. Caspase-3 activity in the presence of chicken extracts. Extracts from
CPVAcrmA::lacZ confluent infections of CAMs harvested at 48 hours (70 pg) (-A-) or
10U of recombinant human caspase-3 (- -)or a mixture of extracts and purified enzyme
(- -) were assayed for the ability to cleave Ac-DEVD-AMC. Enzyme activity was
measured fluorometrically by the ability to cleave peptide substrate Ac-DEVD-AMC and
expressed as fluorescence signal units (FSU).


132
14). Cleavage of Ac-DEVD-AMC is indicative of caspase activity. Cell extracts are
incubated with Ac-DEVD-AMC and the rate of cleavage is determined fluorometrically.
RPV containing functional SPI-2 gene also induces caspase activity, although the rate of
peptide cleavage stabilizes after 9 hours post infection. The rate of cleavage of Ac-
DEVD-AMC in RPVASPI-2::P35 infections was seen to increase after 6 hours post
infection and peaked at 12