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Protein Requirements for the Initiation of Poliovirus Negative-Strand RNA Synthesis

Permanent Link: http://ufdc.ufl.edu/UFE0024753/00001

Material Information

Title: Protein Requirements for the Initiation of Poliovirus Negative-Strand RNA Synthesis
Physical Description: 1 online resource (149 p.)
Language: english
Creator: Spear, Allyn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 3cd, pcbp, poliovirus, replication, rna
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: During infection, poliovirus genomic RNA acts first as a messenger RNA and subsequently serves as a template for RNA replication. Because these processes require exclusive use of the genome, the mechanism by which this transition occurs must be carefully orchestrated. The first step in RNA replication is initiation of negative strand RNA synthesis, however, prior to this initiation a membrane associated viral replication complex must form, requiring key viral and cellular proteins. Using a cell free system, experiments were performed to investigate specific cellular and viral protein requirements for replication complex formation and subsequent initiation of negative strand RNA synthesis. A protein-RNA tethering system was developed to study the involvement of cellular poly(C) binding protein (PCBP) in the initiation of poliovirus negative strand RNA synthesis. The results of these studies showed that PCBP is essential for initiation of negative strand synthesis, and did not require direct RNA binding or multimerization. The critical domain of PCBP was identified and it was shown that multiple PCBP isoforms share this activity. To investigate the viral proteins required for efficient initiation of negative strand synthesis, a series of trans replication reactions were performed. The results of these studies implicate 2BCP3 as the critical cis acting viral protein precursor, essential for membrane associated replication complex formation. This precursor would be severely trans restricted by its association with membranes and its rapid processing, accounting for the dramatic increase in RNA replication efficiency of RNAs which generate the 2BCP3 precursor in cis. Another viral protein precursor, 3CDpro, is also critical for many aspects of viral replication. It has multiple functions, including polyprotein processing, RNA binding, and as the precursor for the polymerase (3Dpol). To investigate the function(s) of 3CDpro involved in the initiation of negative strand RNA synthesis, poliovirus RNAs containing distinct functional mutations within the 3CD coding region were assayed for their ability to be complemented by either wild type or mutant 3CD proteins. The results of these studies indicate the presence of two or more molecules of 3CDpro in the replication complex, and also clearly show that active polymerase must be delivered to this complex in the form of 3CDpro or a larger precursor.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Allyn Spear.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Flanegan, James B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024753:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024753/00001

Material Information

Title: Protein Requirements for the Initiation of Poliovirus Negative-Strand RNA Synthesis
Physical Description: 1 online resource (149 p.)
Language: english
Creator: Spear, Allyn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 3cd, pcbp, poliovirus, replication, rna
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: During infection, poliovirus genomic RNA acts first as a messenger RNA and subsequently serves as a template for RNA replication. Because these processes require exclusive use of the genome, the mechanism by which this transition occurs must be carefully orchestrated. The first step in RNA replication is initiation of negative strand RNA synthesis, however, prior to this initiation a membrane associated viral replication complex must form, requiring key viral and cellular proteins. Using a cell free system, experiments were performed to investigate specific cellular and viral protein requirements for replication complex formation and subsequent initiation of negative strand RNA synthesis. A protein-RNA tethering system was developed to study the involvement of cellular poly(C) binding protein (PCBP) in the initiation of poliovirus negative strand RNA synthesis. The results of these studies showed that PCBP is essential for initiation of negative strand synthesis, and did not require direct RNA binding or multimerization. The critical domain of PCBP was identified and it was shown that multiple PCBP isoforms share this activity. To investigate the viral proteins required for efficient initiation of negative strand synthesis, a series of trans replication reactions were performed. The results of these studies implicate 2BCP3 as the critical cis acting viral protein precursor, essential for membrane associated replication complex formation. This precursor would be severely trans restricted by its association with membranes and its rapid processing, accounting for the dramatic increase in RNA replication efficiency of RNAs which generate the 2BCP3 precursor in cis. Another viral protein precursor, 3CDpro, is also critical for many aspects of viral replication. It has multiple functions, including polyprotein processing, RNA binding, and as the precursor for the polymerase (3Dpol). To investigate the function(s) of 3CDpro involved in the initiation of negative strand RNA synthesis, poliovirus RNAs containing distinct functional mutations within the 3CD coding region were assayed for their ability to be complemented by either wild type or mutant 3CD proteins. The results of these studies indicate the presence of two or more molecules of 3CDpro in the replication complex, and also clearly show that active polymerase must be delivered to this complex in the form of 3CDpro or a larger precursor.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Allyn Spear.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Flanegan, James B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024753:00001


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PROTEIN REQUIREMENTS FOR THE INITIATION OF POLIOVIRUS
NEGATIVE-STRAND RNA SYNTHESIS



















By

ALLYN R. SPEAR


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

2009



































2009 Allyn R. Spear


































To my loving and supportive parents,
who have always taught me to never stop working towards my dreams









ACKNOWLEDGMENTS

I would like to thank Dr. J. Bert Flanegan for the many opportunities he has provided as

well as the extensive scientific freedom he has granted me to pursue many varied avenues of

research. I would like to thank the members of the Flanegan lab for their advice, support,

assistance, and guidance. Brian O'Donnell, Joan Morasco, Nidhi Sharma, Sushma Ogram, and

Jessica Parilla have all contributed in innumerable ways to my scientific development and have

been and always will be my friends. And for the many insightful discussions and suggestions, I

would like to thank all of the members of my committee, Dr. Rich Condit, Dr. David Bloom, Dr.

Linda Bloom, and Dr. Jorg Bungert. I would also like to thank Dr. Rob McKenna for always

being available and willing to listen; you have been like a second mentor to me.

I would like to sincerely thank my parents for their undying support for almost every crazy

thing I have wanted to try in my life, including crossing the country for graduate school.

I would also like to thank my first scientific mentor, Dr. Michael Hoffman. His patience

with me as a young scientist and his extensive mentoring provided me with the foundations of

my scientific training. Dr. Hoffman's commitment to balancing his professional and personal

life, and his dedication to scientific education, continues to be an inspiration to me to this day.

Particularly for her exceptional patience and support during the writing of my thesis, I

would like to thank Zenia Torres. She has given me a brighter outlook on my future than I ever

could have dreamed and I can't imagine my life without her.

Last, but certainly not least, I would like to more directly thank Sushma Ogram for her

camaraderie and support throughout my graduate career. Selfless, caring, and a friend without

condition, Sushma always helped bring me up during the worst of times. I know that I would not

have made it through graduate school without her, and for that I offer my sincerest thanks.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..............................................................................................................4

LIST OF FIGURES ................................... .. .... ..... ................. .8

LIST OF ABBREVIATION S .............. ...................................... .... .................. 10

A B S T R A C T ............ ................... ............................................................ 1 1

CHAPTER

1 BACKGROUND AND SIGNIFICANCE............................................ 13

Poliovirus Pathogenesis and Epidemiology................................ ......................... ........ 13
Poliovirus M molecular Biology.................................................................. ............... 15
A ttachm ent and E ntry ...................... .. .. ......... .. ................................ ............... ... 16
Viral Translation and Polyprotein Processing..... .......... ....................................... 17
Virus-Induced Alteration of Host Cell Environment ............................................... 18
Host Protein Involvement in RNA Replication ........................................... ...............19
N egative-Strand RN A Synthesis ........................................... ..... ....................... 20
P ositive-Strand R N A Synthesis............................................................ .....................2 1
Packaging and Release of Progeny Virions ..................................... ....... ............... 23
C ell-F ree R replication Sy stem ....................................................................... ... .................24

2 M A TER IA L S A N D M ETH O D S ........................................ .............................................28

DNA M manipulation and Cloning Techniques..................................................................... 28
Site-D directed M utagenesis...................................................................... ...................29
T w o-Step P C R ....................................... .............................. ............ 30
Construct Verification and DNA Stock Preparation ...................................................31
cDNA Clones U sed in These Studies .............................................................................. 32
Poliovirus Clones Used in These Studies....................... .... ........... ................. 32
Poliovirus-Based Protein Expression Clones Used in These Studies ...........................37
Bacterial Protein Expression Clones Used in These Studies........................................41
RNA Transcript Preparation and Purification ............................................. ............... 42
Standard Transcription ............................................ .................... ........ 42
Ribozym e Optim ized Transcription ........................................ .......................... 43
5' C apping T transcription ....... .............................................................. .. .... .... ..... 43
H eL a E extract P reparation ............................................................................. .................... 43
S 1 0 P re p a ratio n ...............................................................................................................4 4
IF P rep aratio n ..................... .. ... ... ........................................... 4 5
HeLa S10 Translation-RNA Replication Reactions...........................................................46
RN A Program m ing and Translation.......................................... .......................... 46
PIRC Isolation and RN A Replication......................................... ......................... 47
Analysis of Protein Synthesis by SDS-PAGE................................... ...............48









Analysis of RNA Replication by Denaturing CH3HgOH Gel Electrophoresis......................48
B acterial P rotein E expression .......................................................................... ................... 49
Electrophoretic M obility Shift A ssays......................................................... ............... 50
Riboprobe Synthesis .................................................... ...... ....50
Binding Reactions and Gel Electrophoresis ............................................................. 51

3 POLY(C) BINDING PROTEIN IS REQUIRED FOR EFFICIENT INITIATION OF
POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS...............................................52

Intro du action ................... .......................................................... ................ 52
Results ................... .. .......................................... ....... ......... .55
A Mutation in Stem-loop b of the 5' Cloverleaf Inhibits Negative-strand Synthesis .....55
(M S2)2 Protein-RN A Tethering System .............................. .... .................................. 56
(M S2)2PCBP2 Binds Specifically to 5'CLMS2 RNA ......................................................57
(MS2)2PCBP2 Restores Negative-strand Synthesis on P23-5'CLMS2 RNA ...................58
Deletion Analysis of PCBP2 Using the (MS2)2 Protein-RNA Tethering System ..........58
The Conserved KH3 Domain is Sufficient to Support Negative-strand Synthesis.........60
The Combined KH1-KH2 Domain Fragment Does Not Utilize PCBP Dimerization
to Prom ote N egative-strand Synthesis..................... .................................... .... 61
Multiple PCBP Isoforms Support Initiation of Negative-strand RNA Synthesis ...........62
Not All PCBP Family Members Support Negative-strand synthesis............................64
D iscu ssion ............ .. ........... .. ........................ .... ... .............. .. .. ............. 6 5
Prior Indications of PCBP Involvement in Poliovirus RNA Replication.....................66
(MS2)2 Protein-RNA Tethering Assay Demonstrated that PCBP is Required for
Poliovirus N egative-strand Synthesis ............................................. ...........................67
The Combined KH1 & KH2 Fragment or the KH3 Domain of PCBP2 is Required
for Negative-strand Initiation................... ... ... ... ................................68
A Subset of PCBP Isoforms Support Initiation of Negative-strand RNA Synthesis ......69

4 2BC-P3 IS THE CRITICAL CIS-ACTING VIRAL PROTEIN PRECURSOR
DIRECTING INITIATION OF POLIOVIRUS NEGATIVE-STRAND RNA
S Y N T H E SIS ...................................... ...................................................... 85

Introduction ................... .......................................................... ................. 85
R results ......................... .. .......... ........ ............ ................ ... ......... 86
Efficient PV Negative-strand Synthesis Requires Translation of Viral Template
R N A ................ ...... ........... ..................... .............. .... ........ ...................... 8 7
Template RNA Translation Alone is Not Sufficient to Promote Efficient PV
Negative-strand RNA Synthesis .............................................. ....................... 89
Translation of the 3D Coding Region in cis is Necessary for Efficient PV
N egative-strand Synthesis............................... ............ .... ............................... 90
Translation of the 2BCP3 Protein Precursor in cis is Sufficient for Efficient PV
N egativ e-strand Synthesis......................................... .............................................9 1
D discussion ................... .................................................. .... 92
PV Translation in cis is a Prerequisite for Efficient RNA Replication...........................93
Complete Ribosome Transit Through a Template RNA is Not Sufficient to Promote
High Levels of Negative-strand RNA Synthesis .................................... ...............94









Poliovirus RNA Replication Requires Translation of the 2BC-P3 Precursor in cis .......95
A Model for PV RNA Replication Complex Formation Dependent on cis
Translation of the 2BC-P3 Precursor Polyprotein .................................. ............... 97

5 MUTLIPLE MOLECULES OF THE 3CD VIRAL PROTEIN PRECURSOR
PERFORM DISCRETE FUNCTIONS IN THE INITIATION OF POLIOVIRUS
NEGATIVE-STRAND RNA SYNTHESIS ...................................................103

Introduction ................ ..................... ......... ................ ........... 103
R e su lts ................... ......... ......................... .......... .... ............. ..... ... ... ................ 1 0 5
Mutations Which Prevent the Production of Active 3DP"o are Rescued by 3CDPro.......105
Complementation of 3Dpol Deficient Mutations Requires the Intact 3CDpro
P recu rsor ................................................ ....... ...... ...................... ................ 107
Mutations Which Disrupt 3Cpro/3CDpro Binding to the 5'CL Block RNA
Replication and Affect Polyprotein Processing ...................................................... 108
Complementation of 3Cpro/3CDpro RNA Binding Mutants Requires the Intact
3C D pro P recursor .................................... ........................ ...... .... ... ............109
Complementation Between Two Functionally Distinct 3CDpro Mutants......................110
High Efficiency Complementation of 3C[K12N/R13N] Requires the P3 Precursor.... 112
D isc u ssio n ................... ................................ ........................ ..... ........ ............... 1 1 3
Active 3DP"o is Admitted to the PV Replication Complex in the Form of its
Polym erase-inactive Precursor 3CDPro ................................................... ...............1 13
RNA Binding and Protease Activities of 3CDpro are Functionally Linked............... 114
Multiple 3CDpro Peptides are Present in the PV RNA Replication Complex Used to
Initiate Negative-strand RNA Synthesis.............................. ............... ............... 115
The 3CDpro Bound to the 5'CL is Admitted to the PV Replication Complex in the
Form of its Precursor P3 ..................................................................................... 116

6 SUMM ARY AND CONCLUSIONS........................................................ ............. 126

The Role of PCBP in the Initiation of Poliovirus Negative-strand Synthesis ......................126
The (MS2)2 Protein-RNA Tethering System: Virus-Host Interaction .......................127
The (MS2)2 Protein-RNA Tethering System: Host Protein Function..........................127
The Role of Viral Protein Precursors in the Initiation of PV Negative-strand Synthesis.....128
Modeling Formation of the PV RNA Replication Complex .............. ................128
Close Coupling of the Viral Life-Cycle Ensures Viral Fitness ................................... 129

L IST O F R E F E R E N C E S ..................................................................................... ..................13 1

BIOGRAPHICAL SKETCH ............................................................. ...........149









LIST OF FIGURES


Figure p e

1-1 Poliovirus genome organization and polyprotein processing cascade.............................25

1-2 Poliovirus life-cycle.................... ......................... ........... 26

1-3 Genomic circularization models for PV translation and replication...............................27

3-1 Diagrams of the wild-type and mutant 5' cloverleaf. ................. ...............72

3-2 The C24A mutation affects negative- but not positive-strand RNA synthesis ................73

3-3 Schematic of the (MS2)2 protein-RNA tethering system.. ..............................................74

3-4 The 5'CLMS2 binds (MS2)2 fusion proteins but does not bind PCBP2 ...........................75

3-5 The (MS2)2PCBP2 fusion protein restores negative-strand synthesis of a 5'CLMS2
R N A tem plate .............................................................................76

5-6 Identification of the functional domains within PCBP2 that restore negative-strand
RNA synthesis of a 5'CLMS2 template RNA.. .......................... ...... ............................ 77

3-7 Levels of protein synthesis observed in the (MS2)2 protein-RNA tethering replication
reactions .................................................................................78

3-8 Characterization of the KH3 domain using the (MS2)2 protein-RNA tethering
sy stem ................... .......................................................... ................ 7 9

3-9 The (MS2)2 fusion proteins are evenly expressed, stable, and bind to 5'CLMS2 with
sim ilar affin ity ............................. ............................................................. ............... 8 0

3-10 The ability of the combined KH1/2 domains to restore negative-strand synthesis does
not require the m ultim erization dom ain....................................... .......................... 81

3-11 PCBP1, PCBP2, and PCBP2-KL restore negative-strand synthesis to similar levels in
the (MS2)2 protein-RNA tethering system................ ............................82

3-12 PCBP4/4A, but not PCBP3 or hnRNP K, restores negative-strand synthesis in the
(M S2)2 protein-RNA tethering system .. ................................... ... ........ ..................... 83

3-13 All PCBP family proteins, except hnRNP-K, bind to the PV 5'CL. ................................84

4-1 Translation of a PV RNA template is a prerequisite for efficient negative-strand
synthesis.......... .............................. ................................................98

4-2 Physical ribosome transit of a template RNA is not sufficient to promote efficient
initiation of negative-strand synthesis.. ........................................................ ......... 99









4-3 Translation of 3D or a 3D precursor is required in cis for efficient initiation of
negative-strand synthesis. ....................................................................... ...................100

4-4 Efficient initiation of negative-strand synthesis requires translation of 2B or a 2B
p recu rsor in cis............................................................................................ . 10 1

4-5 Poliovirus RNA replication requires translation of the 2BC-P3 polyprotein precursor
in cis.. ............... ......... ........................................................................ .......... ...... 10 2

5-1 Mutations which prevent the generation of active 3DP"o block RNA replication. ...........118

5-2 Viral Precursor 3CDPro complements both 3D[G327M] and 3CD[PM] in trans. ...........119

5-3 Complementation of 3D[G327M] or 3CD[PM] requires the intact 3CDPro precursor. ...120

5-4 Mutations which disrupt 3Cpro/3CDPro binding to the 5'CL block RNA replication....... 121

5-5 Complementation of 3C[K12N/R13N] or 3C[R84S] requires the intact 3CDpro
precursor ................................................................................122

5-6 Schematic of trans complementation using two functionally distinct mutations in
3CDpr .......... ........................................ ................... ............123

5-7 Two functionally distinct 3CDPro mutants can complement each other in trans ...........124

5-8 Complementation of a 3CDPro RNA binding mutant is more efficient when P3 is
provided in trans ............................................ ..................................... 12 5









LIST OF ABBREVIATIONS

ATP Adenosine 5' triphosphate

cDNA Complimentary DNA

CTP Cytidine 5' triphosphate

DNA Deoxyribonucleic Acid

GTP Guanosine 5' triphosphate

GuHCl Guanidine Hydrochloride

HeLa Human cervical carcinoma cell line

IF Initiation factors (Ribosomal salt wash protein preparation)

IRES Internal ribosomal entry site

kDa Kilodalton

NTP Nucleoside 5' triphosphate

NTR Non-translated region

PABP Poly(A) binding protein

PCBP Poly(C) binding protein

PIRC Pre-initiation replication complex

pol Polymerase

poly(A) Polyadenosine 5' triphosphate

pro Protease

RNA Ribonucleic Acid

S10 Supernatant from a 12,000 x g centrifugation

UTP Uridine 5' triphosphate

vRNA Virion RNA

wt Wild type









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

PROTEIN REQUIREMENTS FOR THE INITIATION OF POLIOVIRUS
NEGATIVE-STRAND RNA SYNTHESIS

By

Allyn R. Spear

August 2009

Chair: James Bert Flanegan
Major: Medical Sciences Biochemistry and Molecular Biology

During infection, poliovirus genomic RNA acts first as a messenger RNA and

subsequently serves as a template for RNA replication. Because these processes require

exclusive use of the genome, the mechanism by which this transition occurs must be carefully

orchestrated. The first step in RNA replication is initiation of negative-strand RNA synthesis,

however, prior to this initiation a membrane associated viral replication complex must form,

requiring key viral and cellular proteins. Using a cell-free system, experiments were performed

to investigate specific cellular and viral protein requirements for replication complex formation

and subsequent initiation of negative-strand RNA synthesis.

A protein-RNA tethering system was developed to study the involvement of cellular

poly(C) binding protein (PCBP) in the initiation of poliovirus negative-strand RNA synthesis.

The results of these studies showed that PCBP is essential for initiation of negative-strand

synthesis, and did not require direct RNA binding or multimerization. The critical domain of

PCBP was identified and it was shown that multiple PCBP isoforms share this activity.

To investigate the viral proteins required for efficient initiation of negative-strand

synthesis, a series of trans replication reactions were performed. The results of these studies

implicate 2BC-P3 as the critical cis-acting viral protein precursor, essential for









membrane-associated replication complex formation. This precursor would be severely

trans-restricted by its association with membranes and its rapid processing, accounting for the

dramatic increase in RNA replication efficiency of RNAs which generate the 2BC-P3 precursor

in cis.

Another viral protein precursor, 3CDpro, is also critical for many aspects of viral

replication. It has multiple functions, including polyprotein processing, RNA binding, and as the

precursor for the polymerase (3DPo'). To investigate the functions) of 3CDpro involved in the

initiation of negative-strand RNA synthesis, poliovirus RNAs containing distinct functional

mutations within the 3CD coding region were assayed for their ability to be complemented by

either wild type or mutant 3CD proteins. The results of these studies indicate the presence of

two or more molecules of 3CDPro in the replication complex, and also clearly show that active

polymerase must be delivered to this complex in the form of 3CDpro or a larger precursor.









CHAPTER 1
BACKGROUND AND SIGNIFICANCE

Poliovirus Pathogenesis and Epidemiology

Poliovirus (PV), the causative agent of poliomyelitis, is a member of the family

Picornaviridae, in the genus enterovirus. The viruses of the family Picornaviridae are small

(25-30 nm diameter), non-enveloped, icosahedral (T=3), positive-sense ssRNA viruses, and can

cause a variety of human diseases, including meningitis, encephalitis, poliomyelitis, pancreatitis,

myocarditis, rhinitis, and hepatitis. PV is spread via the fecal-oral route, typically by ingestion

of contaminated food or water. The primary site of replication is in the mucosal lining of the

oropharyngeal and intestinal tract, either in epithelial or lymphoid cells, and during acute

infection, virus is shed at high levels in the feces of infected individuals (171). Infectious virus

is very stable in the environment, persisting in contaminated groundwater for 3-6 weeks or more

(81). After primary infection in the alimentary tract, PV may enter the central nervous system by

one of two routes, either by infection of a peripheral nerve and subsequent retrograde intraaxonal

transport or by crossing the blood-brain barrier following viremia (171). Paralytic poliomyelitis

occurs in 0.5-1% of infected individuals and is a direct result of the death of motor neurons in the

spinal cord and/or motor cortex caused by infection and lysis from PV infection. Due, in part, to

its extensive study following the poliomyelitis epidemics in the mid 20th century, PV has become

the prototypical member of the Picornaviridae for studying the molecular mechanisms of viral

replication.

Although paralytic polio has probably affected mankind throughout much of recorded

history, the epidemics of infantile paralysis in the early to mid 20th century are what most people

associate with the concept of poliomyelitis. It was the widespread, devastating nature of this

disease that spurred scientists around the world to further characterize the infectious agent and









develop effective vaccines. The success of the vaccination campaign, and the lack of an animal

reservoir, led the World Health Organization (WHO) to attempt global eradication set to be

completed by the early 21st century. In the course of the 20 year campaign, the cost of the

Global Polio Eradication Initiative (GPEI) has exceeded six billion dollars, and despite

tremendous progress, poliomyelitis has recently been on the rise in areas in which poliovirus

remains endemic (3, 4, 177). The Americas were the first region certified polio-free by the

WHO in 1994, followed by the Western Pacific in 2000, and Europe in 2002 (69).

Unfortunately, a few regions in Africa and Asia have resisted the best efforts by the GPEI,

specifically Nigeria, Afghanistan, Pakistan, and India, and all cases of wild poliovirus

transmission since 2006 can be traced back to virus export from one of these four countries (3).

The lack of success in these regions can be attributed to multiple factors, not the least of which

are inhospitable socio-political climates, adverse geography, and an as yet unexplained variance

in the host immune response to vaccination (69). In Nigeria, as a result of rumors of

vaccine-induced infertility, vaccination coverage dropped dramatically in 2002-03, leaving an

even larger proportion of the population susceptible. Additionally, due to apparent success in

northern India, aggressive vaccination campaigns were scaled back beginning in 2002. By 2006,

20 countries that had previously been polio-free reported importation of Nigerian polioviruses, 3

polio-free countries reported cases of Indian poliovirus, and worldwide cases had risen to over

2000 (1-3, 174). This number has not changed significantly since 2006 and worldwide incidence

of poliovirus remains between 1000 and 2000 cases per year, with exportation of poliovirus from

endemic countries remaining a serious concern. The WHO and GPEI has recently recommitted

itself to the campaign, setting new deadlines for polio eradication: India, Afghanistan, and

Pakistan by 2010, Nigeria by 2011 (177). The prospect of nearing eradication presents new









challenges and generates some interesting scientific questions: Since the use of oral polio

vaccine (OPV) has an associated risk of causing vaccine derived poliomyelitis, how will the

OPV be phased out and the inactivated polio vaccine (IPV) be phased in? Will the immune

responses and vaccine coverage attainable using the IPV be sufficient to protect the world's

population? Will we ever be able to stop vaccination for poliovirus? In the process of

discussing these issues, the field has determined that the development of anti-polioviral drugs

would be a significant benefit to public health, particularly during the transition into a "polio-free

world" (reviewed in 59). To date, there have been no such drugs that have shown any clinically

promising results, indicative of a need for a better understanding of the molecular biology of

poliovirus replication and the identification of new potential drug targets.

Poliovirus Molecular Biology

The PV RNA genome (Figure 1-1A) is -7.5 kilobases long, uncapped, and covalently

linked to a 22 amino acid viral protein 3BVPg (viral protein genome-linked; VPg) via a

phosphodiester linkage between 04 on tyrosine 3 of VPg and the 5' phosphate on the genome

terminal uridine (10, 120, 179). This VPg moiety is quickly removed by a cellular unlinking

activity (11) and is not required for viral translation (166). Bases 1-89 of the 5' non-translated

region (NTR) of PV genomic RNA form a cis-acting structure known as the 5' cloverleaf (5'CL)

which is required for genomic replication (13, 26, 93, 126, 196), RNA stability (144), and

optimal translation (78, 158, 187; Ogram et al., unpublished results). More recently, a conserved

cytidine-rich sequence (poly(C) tract) in the 12 nucleotides adjacent to the 5'CL was identified

and shown to be required for RNA replication (205). The remainder of the 5'NTR contains a

highly structured region which functions as a type-1 internal ribosomal entry site (IRES), driving

cap-independent initiation of translation (97, 164). Downstream of the IRES is a single open

reading frame encoding viral capsid and non-structural proteins as a single large polyprotein









which is processed by viral proteases (Figure 1-1B) (113). The 3' NTR of PV genomic RNA

contains additional cis-acting structures which, while not absolutely essential (201), are required

for efficient RNA replication (100, 138, 168). Finally, the genomic RNA terminates in a poly(A)

tail of heterogeneous length (-90 nts) (218) which enhances IRES translation (32, 136, 192, 193)

and is required for PV RNA infectivity (190).

Attachment and Entry

The PV lifecycle initiates with the attachment of virions to the Ig-like cell surface receptor

CD155/Pvr (Figure 1-2, Step 1)(111, 134)via surface residues on the PV capsid in a region

known as the "canyon" which surrounds a vertex on the icosahedral particle (87). The capsid

then undergoes conformational changes whereby the myristoylated N-terminus of VP4 and the

hydrophobic N-terminus of VP 1 insert into the cell-membrane, forming a pore structure and a

membrane anchor (41, 42, 75). Recent work by Brandenburg et al. showed that PV

internalization into HeLa cells by endocytosis utilizes a clathrin-, calveolin-, and flotillin-

independent pathway, and that subsequent uncoating and genome release occurs just inside the

plasma membrane at the cell periphery (39). The study further showed that PV entry and

uncoating required ATP, the actin cytoskeleton, and tyrosine-kinase activity. Work by Coyne et

al. showed that PV entry into polarized brain vascular endothelial cells did require calveolin and

dynamin, indicating different mechanisms of entry into different cell types (63). More

interestingly, Coyne et al. also showed that the interaction of PV with CD155 triggered a

signaling cascade involving tyrosine phosphorylation and dramatic rearrangements in the actin

cytoskeleton which were also essential for viral entry and uncoating. Together, these studies

indicate that receptor binding by PV serves a purpose beyond attachment, in the induction of

cellular signaling cascades critical to establishing a cellular environment conducive to infection.









Viral Translation and Polyprotein Processing

Cap-independent translation of the genomic RNA is the first intracellular step in the viral

life cycle (Figure 1-2, Step 2). Because viron RNA (vRNA) does not contain a 5' 7mG cap

structure, it cannot undergo canonical cap-dependent ribosomal scanning and translation as

would a typical eukaryotic mRNA. Instead, picornaviruses utilize a complex series of RNA

secondary structures in their 5'NTR called an IRES to recruit and position ribosomes in a

cap-independent manner (97, 164). The PV IRES is sub-classified as a type-1 IRES based on

structural characteristics, and shares homology with the IRESes of other entero- and rhinoviruses

(99). Like other type-1 IRESes, the PV IRES binds a set of host cell proteins to assist in RNA

folding and ribosome recruitment, including the La autoantigen, poly(C) binding protein 2

(PCBP2), SRp20, polypyrimidine tract binding protein (PTB), upstream-of-N-ras protein (unr),

and eIF4G (90, 131). In addition to these co-factors, certain RNA elements within the IRES

presumably act as a scaffold for the assembly of ribosomal subunits to facilitate efficient

translation initiation.

Eukaryotic cells have evolved a mechanism to further optimize the cap-dependent

translation of their own mRNA by inducing a 5'-3' circularization via interactions between cap

binding protein (eIF4G) and poly(A) binding protein (PABP) bound to the 3' poly(A) tail (212).

In turn, there is a growing body of evidence (reviewed by (131)), as well as a significant amount

of unpublished data from our lab, that indicates a similar enhancement strategy in use by PV

(Ogram et al., unpublished results). Because the PV genome has VPg linked to its 5' end rather

than a 5' 7mG cap structure, it is likely that an alternative protein bridge mediates the 5'-3'

circularization of PV genomic RNA (Figure 1-3). This presumably involves interactions

between PCBP bound to the 5'CL and PABP bound to the 3' poly(A) tail, although additional









and/or alternative interactions have been proposed, including as yet unidentified cellular proteins

(131).

Downstream of the IRES is the single open reading frame which encodes the

approximately 2200 amino acid viral polyprotein. This large protein product is processed by the

viral proteases 2A (2Apro) and 3C/3CD (3Cpro/3CDPro) and the resultant polyprotein cleavage

cascade is depicted in Figure 1-1B (119, 155, 156). Initial polyprotein processing occurs

co-translationally at the boundary between the structural and non-structural proteins by 2Apro

(206), and all subsequent cleavage events (except at the capsid protein VP4-VP2 junction) are

mediated by the other viral protease or its immediate precursor, 3CPro/3CDpro (113, 155). The

cleavage at the VP4-VP2 junction is catalyzed by amino acids in VPO and is induced upon RNA

packaging following final capsid assembly (15, 27, 155). Processing of the non-structural

replication proteins occurs by two distinct pathways determined by the site of primary cleavage

by 3Cpro/3CDpro (119). If the primary cleavage occurs at the 2C-3A junction, processing of the

resultant P2 and P3 precursors proceeds in the soluble phase, and proceeds very slowly.

However, with 3-fold higher frequency, the primary cleavage occurs at the 2A-2B junction,

shunting the resultant 2BC-P3 precursor into the rapid processing membrane associated pathway.

This is of particular importance given that PV infection induces dramatic membrane

rearrangements which are essential for PV replication (44, 45).

Virus-Induced Alteration of Host Cell Environment

The remarkable rearrangement of intracellular membranes observed during PV infection is

dependent on PV translation and results in the formation of rosette-like vesicles to which PV

replication complexes localize (44, 45, 70). It has been shown that viral protein precursor 2BC

and viral protein 2CATPase are responsible for these rearrangements (9, 20, 50). In addition, 2BC

and 2CATPase, as well as 3AB, have been shown to affect membrane permeability and









nucleo-cytoplasmic trafficking (8, 30, 31, 84, 117). The collective effect of these activities

increases the cytoplasmic availability of nuclear factors utilized in PV replication and also

generates the ideal membrane microenvironment which is essential for replication complex

assembly and RNA replication.

In addition to the alteration of host cell membranes, PV also induces a global

down-regulation of host-cell translation and transcription. Beyond their role in PV polyprotein

processing, 2Apro and 3CDpro/3Cpro are also responsible for the comprehensive proteolytic attack

on critical cellular proteins and processes that causes this host shut-off The host cell

translational machinery is primarily disabled by 2Apro cleavage of eIF4GI/II, an essential

component of the cap binding complex eIF4F (72, 82, 125). In addition, both 2Apro and

3CDPro/3Cpro cleave poly(A) binding protein (PABP), another cellular protein involved in

stimulating cap-dependent translation (101, 116). Transcriptional machinery is also

proteolytically inactivated by 2Apro cleavage of TATA-binding protein (TBP) (214), as well as

3CDpro/3Cpro cleavage of TBP (55), cAMP responsive element binding protein (CREB) (215),

Oct-1 (216), and multiple other transcription factors specific for RNA polymerase II (181), and

RNA polymerase III (54, 184). The net effect of these cleavage events is a shut-off of over 95%

of host cell gene expression by three hours post-infection (102).

Host Protein Involvement in RNA Replication

Both PCBP and PABP, discussed above relative to PV translation, have also been shown

to be involved in other aspects of PV replication. As previously discussed, the PV genome

contains an essential RNA structure at the 5' end, so-termed the 5' cloverleaf (5'CL). PCBP

binding to a stem-loop within this structure has been shown to be involved in RNA replication

(12, 13, 158, 209), as well as in stabilizing PV RNA (144). In addition to its association with the









5'CL, PCBP also binds to a conserved cytidine-rich sequence adjacent to the 5'CL and this

interaction was also shown to be required for RNA replication (205).

At the opposite end of the PV genome, the 3' poly(A) tail is of sufficient length to bind

cellular PABP. It has been established that the poly(A) tail plays a role in stability, translation,

and RNA replication, most likely as a result of its interaction with PABP (93, 182, 190; Ogram et

al., unpublished results) Moreover, Silvestri et al. showed that the PABP/poly(A) requirement

was specific for the initiation of negative-strand RNA synthesis (186).

Negative-Strand RNA Synthesis

The transition from PV translation to genomic RNA replication, as with all positive-strand

RNA viruses, must initiate with the synthesis of anti-genomic negative-strand RNA (Figure 1-2,

Step 3. As discussed previously, membrane vesicles are essential for RNA replication since the

cytoplasmic surface of these membranes is the site of replication complex assembly (34, 51). In

addition to membrane vesicles, RNA replication requires viral proteins as well as some forms of

their precursors. Of all the viral proteins and precursors described to date, it has been shown that

2B, 2CATPase, 3AB, VPg(3B), 3Cpro/3CDPro, and 3DP"o are essential for RNA replication (12, 175,

213). Additional data generated from complementation analysis has indicated that optimal

replication may require larger precursor forms of one or more of these essential proteins (104,

124, 204). The precise functions of 2B and 2CATPase in RNA replication are unknown, however,

negative-strand initiation is blocked by millimolar concentrations of guanidine HC1 (GuHC1) and

GuHCl resistance mutations map to 2CATPase (23, 170). The requirement for 3AB may arise from

a need to provide the essential VPg protein primer to the replication complex in a membrane

associated precursor form (46). Data from our lab has suggested that the RNA dependent-RNA

polymerase (3Dpol) must also be delivered to the replication complex in the form of its precursor

3CDpro, prior to processing and negative-strand initiation (25).









The polymerase initiates negative-strand synthesis at the extreme 3' end of the viral RNA,

using the poly(A) tail as a template and VPg as a protein primer. In addition to the presence of

the PABP-poly(A) RNP complex, additional interactions occur on the adjacent 3'NTR, including

recruitment of 3CDpro/3DP"o and 3AB/VPg to provide the active polymerase and protein primer,

respectively (89). Interestingly, despite its location at the opposite side of the genome, an RNP

complex formed on the 5'CL has also been shown to be essential for RNA replication (12, 13).

This complex consists of both cellular PCBP as well as the viral 3CDpro (12, 158, 209). The

involvement of both distal and proximal RNA elements in negative-strand initiation lead our lab

and others to propose the formation of a 5'-3' circular RNP complex, driven by protein-protein

interactions between RNP complexes at the 5'CL and 3'NTR/poly(A) tail (Figure 1-3) (26, 93,

126, 196). Potential bridging interactions could involve a PCBP and PABP interaction, as has

been shown to occur on the 3' end of a-globin mRNA (49, 210). A 3CDPro-3CDPro interaction

could also either drive or augment circularization, since the viral precursor has been shown to

bind to both 5' and 3' ends (12, 89), and interaction surfaces were identified in the crystal

structure of 3DP"o and 3CDP"o (86, 130, 153) Regardless of which is the critical interaction, the

formation of the complete circular RNP complex represents the last pre-replication state of viral

genomic RNA. Once the complex has formed and subsequent processing of any necessary

protein precursors is complete, 3DP"0 initiates RNA replication via uridylylation of VPg on the

poly(A) tail. This would then be followed by elongation of negative-strand RNA, generating a

full-length double-stranded replicative form (RF) RNA.

Positive-Strand RNA Synthesis

Poliovirus RNA replication is highly asymmetric, generating 10-100 molecules of VPg

linked positive-strand RNA for every one negative-strand template synthesized (Figure 1-2,









Step 4) (150, 207). The requirements for the initiation of positive-strand synthesis differ

significantly from those for negative-strand initiation.

First among these differences is the requirement for pre-uridylylated VPg (VPgpUpU) as a

primer for positive-strand elongation (141, 143). The synthesis of VPgpUpU occurs on an RNA

hairpin template in the 2CATPase coding region termed the cis replication element or cre(2C)

hairpin, and requires VPg, UTP, 3DP"o, 3CDpro, PCBP and the 5'CL (126; Sharma et al.,

unpublished results). The uridylylation of VPg is templated by the first of three conserved

adenosines in the loop of the hairpin, and addition of the second uridyl residue is accomplished

via a slide-back mechanism (161, 176). Interestingly, the cre(2C) dependent VPg uridylylation

reaction is inhibited by GuHCl (126, 141), indicating the involvement of 2CATPse, even though

positive-strand synthesis per se is not sensitive to GuHCl inhibition (23).

The protein requirements for positive-strand initiation are also different from those

observed for negative-strand synthesis. Despite GuHCl insensitivity, 2CATPase has been shown to

bind specifically to the 3' end of the negative-strand, indicating a possible role in positive-strand

synthesis (19). Data from the Semler lab has established the specific binding of cellular protein

hnRNP C to the 3' end of negative-strand RNA, and have shown it to be required for RNA

replication (40, 178). Sequences at the 3' end of PV negative-strand RNA, which correspond to

a potential hnRNP C binding site, have also been shown to be essential for positive-stand

synthesis (183). Taken together, these data suggest a mechanism of positive-strand initiation

whereby hnRNP C, 3DP"0, VPgpUpU, and possibly 2CATPase form an RNP complex at the 3' end

of the negative strand RNA (or RF RNA) to promote multiple sequential rounds of

positive-strand RNA synthesis.









Packaging and Release of Progeny Virions

When sufficient PV capsid protein and genomic RNA have been synthesized, virion

assembly begins (Figure 1-2, Step 5). There is no known packaging signal or sequence

requirement for encapsidation of PV RNA, however through exhaustive study of defective

interfering (DI) particles and induced genomic deletions, it has been determined that the capsid

coding region is not required for vRNA encapsidation (52, 115, 148). Additional studies have

shown that although not essential, PV IRES sequences do enhance encapsidation (103). There

also appears to be a very strict discrimination in RNA polarity, since packaging of

negative-sense RNA is undetectable (150). In addition to sequence requirements, all packaged

RNAs must be VPg linked (149), and there also appears to be a tight coupling between active

RNA replication and encapsidation of nascent vRNA (152). Once the RNA is encapsidated, VPO

undergoes processing to generate VP4 and VP2, mediated by catalytic residues in VP2 and

activated by the presence of RNA, which results in formation of the final infectious virus particle

(15, 27, 91, 94).

Release of viral particles from infected cells can occur by multiple mechanisms, including

programmed cell death (203), cytopathic effect (CPE) induced lysis (7), and

autophagosome-mediated exit without lysis (AWOL) (195). Although poliovirus infection

induces pro-apoptotic programs, the programs are quickly suppressed by viral proteins (203). In

fact, it is the interaction of PV with its receptor (CD 155) that induces c-Jun NH2-terminal kinase

(JNK) activation, and ultimately this activation overcomes viral protein mediated suppression

and triggers cell death via Bax-dependent mitochondrial dysregulation, cytochrome c release,

and activation of the apoptotic caspase cascade (7, 16). Alternatively, in the presence of

inhibitors of apoptosis, Agol et al. showed that the CPE caused by PV infection (e.g. membrane

rearrangements, increase in nuclear permeability) were sufficient to induce lysis of the host cell









and release of viral particles (7). The release of small amounts of infectious virus has also been

observed in the absence of cell lysis, and Taylor et al. has recently shown that this is a

consequence of viral subversion of the cellular autophagy pathway resulting in the delivery of

small pockets of virus-laden cytoplasm to the extracellular space (195). In all cases, these newly

formed viral particles, following release or lysis of the host cell, can now either spread to infect

neighboring cells, or be shed into the environment to await a new host.

Cell-Free Replication System

Shortly after the isolation and establishment of the HeLa cervical carcinoma cell line in

1951, HeLa cells were widely used to passage and study poliovirus (5, 194). A major

breakthrough in PV molecular biology was the cDNA cloning and sequencing of the PV genome

by Racianello and Baltimore in 1981 (172, 173). This was followed by another significant

advance ten years later, when Molla, Paul and Wimmer successfully generated infectious

poliovirus de novo using a cell-free replication system (140). Further optimization and

characterization of this system by our lab and others has produced the cell-free HeLa S10

translation-replication system in use today (21, 22, 24). This system permits us to uncouple the

otherwise intertwined processes of translation and replication, allowing us to finely dissect the

molecular mechanisms of these events, while still accurately recapitulating in vivo viral

replication. Recent developments involving the use of ribozyme generated authentic 5' ends on

transcript RNAs have allowed us to further dissect RNA replication and examine the molecular

biology and genetics of negative-strand and positive-strand synthesis separately (92, 141). Using

this system, we have begun to identify the viral and cellular proteins required for the initiation of

poliovirus negative-strand RNA synthesis.









A.
5' Clovereaf

VPg
poly(C)




B.


il I


Polyprotein
.-- Capsid Non-Structural

- -


S P23


VPO VP31 VPI

VP2
VP4


Membrane
SAssociated


2BC-P3


Soluble


P2


2BC


2B1 2C


3A
3A VPg


3AB
2BC .


3D""


Figure 1-1. Poliovims genome organization and polyprotein processing cascade. A) PV genomic
RNA is covalently linked to VPg at its 5' end, and contains multiple cis acting RNA
elements, including the 5' cloverleaf (5'CL), poly(C) tract, IRES, cre(2C) stem-loop,
and 3'NTR/poly(A) tail. B) PV translates a single large polyprotein which is
processed by viral proteases 2Apro (f) and 3Cpro/3CDpro (*). Primary cleavage of the
P23 precursor at the 2A-2B junction occurs with 3-fold higher efficiency than
cleavage at the P2-P3 junction. This initial processing event determines if the
subsequent processing occurs in membrane-associated or soluble compartments.


3'NTR


(A)811


P3

3AB

*1










CD155
-I o


Cell Membrane


--AAAAAAAAAAAAAAAt
----------------r rrrrit Hit Iitrrite


Figure 1-2. Poliovirus life-cycle. PV binds its cellular receptor CD155, undergoes
internalization by endocytosis, and releases its genome into the cytoplasm at the cell
periphery (Step 1). Upon release, translation factors and ribosomes assemble on the
IRES and viral protein synthesis occurs (Step 2). Using these newly synthesized
proteins, the viral replication complex is formed and the 3D polymerase generates a
new VPg-linked negative-strand RNA (Step 3). This dsRNA intermediate is then
used as a template for multiple rounds of VPg-UU primed positive-strand synthesis
(Step 4). When sufficient RNA and protein synthesis has occurred, nascent
positive-strand genomic RNAs are packaged by the viral capsid proteins and these
progeny virions are released upon apoptosis of the host cell (Step 5).


"ooo;


'j;Q













Genome
Circularization


Translation and
Ribosome Reloading


Initiation of
Negative-Strand
RNA Synthesis


Figure 1-3. Genomic circularization models for PV translation and replication. Prior to viral
protein synthesis, interactions between PCBP and PABP circularize PV genomic
RNA to facilitate ribosome reloading and enhance translation. Following viral
protein synthesis, replication protein precursors 3CDpro and 3AB would be recruited
to the 5'CL and 3'NTR, undergo processing, and initiate VPg-primed negative-strand
RNA synthesis.









CHAPTER 2
MATERIALS AND METHODS

DNA Manipulation and Cloning Techniques

All restriction enzymes, as well as the Klenow fragment of T4 DNA polymerase, used in

these studies were obtained from New England Biolabs unless otherwise noted. Restriction

digests were performed according to manufacturer's protocols, and when double digests were

required they were performed sequentially unless optimal conditions were available for

simultaneous digests. Standard PCR reactions were carried out according to the manufacturer's

suggested protocols using either TrueFidelity DNA polymerase (Continental Lab Products),

PfuUltra Fusion II DNA polymerase (Stratagene), Accuprime Pfx DNA polymerase (Invitrogen),

or Phusion DNA polymerase (New England Biolabs). Rapid purification of PCR products for

direct restriction digest was performed using QiaQuick PCR Cleanup kit from Qiagen. Gel

purification of PCR fragments or restriction enzyme digested DNA was performed using the

GeneClean II Spin Kit from BiolOl. For gel purification, DNA fragments were resolved on

SeaKem GTG agarose (formulated for gel purification) from Cambrex, visualized by ethidium

bromide staining on a low intensity UV transilluminator, and appropriate bands were excised

using a scalpel. All vector DNAs, where required, were dephosphorylated using Shrimp

Alkaline Phosphatase (SAP) from Roche Applied Science. It is critical that dephosphorylation

be performed after gel purification, as this will reduce vector background to near zero.

Dephosphorylation reactions were performed by adding SAP to 10% of the total reaction volume

and using the provided 10X dephosphorylation buffer [500 mM TrisHCl (pH=8.5), 50 mM

MgC12,]. Following a 1 h incubation at 370C, the SAP was inactivated by incubation at 650C for

15 min. Vector and insert DNA fragments were quantitated by agarose gel electrophoresis and

ethidium bromide visualization of each fragment versus the 1 kb or 100 bp DNA ladder (New









England Biolabs). Ligations were performed using T4 DNA ligase obtained from Promega. All

ligations utilized the provided 10X ligase buffer [300 mM TrisHCl (pH=7.8), 100 mM MgCl2,

100 mM DTT, 10 mM ATP] and contained 1 Unit/pL T4 DNA ligase. All reactions contained a

total of 5 ng/iL DNA (vector + insert), however, for sticky-sticky and sticky-blunt ligations, a

ratio of 1:3 (vector:insert) was used, whereas for blunt-blunt ligations, a ratio of 1:1

(vector:insert) was used. For sticky-sticky ligations, reactions were incubated at room

temperature for 1 h or more prior to transformation, and for sticky-blunt or blunt-blunt ligations,

reactions were incubated at room temperature for at least 12 h or more prior to transformation.

Site-Directed Mutagenesis

Site-directed mutagenesis was performed using a procedure based on that of the

Stratagene's QuikChange site-directed mutagenesis kit. Briefly, two complementary mutagenic

primers are designed which contain the desired mutations flanked on either side by 10-15 nts of

non-mutagenic complementary sequence. Using these primers, the appropriate template, and

PfuTurbo DNA polymerase (Stratagene), PCR reactions were performed where the elongation

times were extended to allow complete transit around the circular plasmid DNA template. After

18 PCR cycles, 20 Units of DpnI was added to the reaction and incubated for 1 h at 370C to

digest all methylated input DNA template. Following digestion, 1 [iL of this reaction was

transformed into XL-1 Blue competent cells (Stratagene). Resultant ampicillin resistant colonies

were screened by DNA mini-prep and restriction digest and sequencing. Once the sequence of

the mutated region was verified, a restriction fragment within the sequenced region, containing

the mutation, was transferred back into the parent vector background to prevent accumulation of

secondary vector mutations that could potentially arise during PCR of the entire plasmid.









Two-Step PCR

Two-step PCR was performed to induce mutations (two-step mutagenic PCR) as well as to

fuse two DNA sequences (two-step semi-overlapping PCR). For mutagenic PCR, two

complementary mutagenic primers were designed which contain the desired mutations flanked

on either side by 15-20 nts of non-mutagenic complementary sequence. Additionally, two

outside primers were designed, one 5' of the desired mutation and one 3' of the desired mutation.

For convenience, these primers were each 300-1000 bases away from the position of the

mutatgenic primers. It was also essential that these outside primers also encompassed unique

restriction enzyme sites for reintroduction of the mutated fragment. Two first step PCRs were

performed using standard PCR conditions and enzymes described above. These PCRs both

utilized the same template DNA, however one contained the primer pair to generate the 5'

product and the other reaction contained the primer pair to generate the 3' product. Here, both

products share the entire mutagenic primer sequence (i.e. the 3' end of one product is fully

complementary to the 5' end of the other). These first step PCR products were then gel purified

and quantitated using methodology described above. The second step PCRs contained 10-20 ng

of each first step PCR product and only the 5' and 3' outside primers. Here, the first step

products are added in place of plasmid template DNA and the mutations are already present in

these templates, so mutagenic primers are no longer necessary. All second step PCRs used only

PfuUltra Fusion DNA polymerase (Stratagene) or Phusion DNA polymerase (New England

Biolabs) as these were empirically determined to generate the highest product yield with the

lowest extraneous background amplification. The second step PCR product was a result of

priming of one first step PCR product on the other, followed by amplification of this combined

product by the outside primer pair. The second step PCR product was purified using the

QiaQuick PCR purification kit and the resultant DNA was digested using the restriction enzymes









whose sites flanked the induced mutation. The restriction fragment of the second step PCR

product, which contained the desired mutation, was then cloned into the corresponding sites of

the parent plasmid DNA.

In cases where two DNA sequences needed to be fused, two-step semi-overlapping PCR

was used. To do this, a sequence map of the final desired (fused) sequence was generated. Two

complementary primers were then designed such that the primer pair equally spanned the

junction between the two fused sequences. This resulted in the generation of two primers which

each contained equal halves of two distinct sequences. As with two-step mutagenic PCR

described above, two additional outside primers were also required, however these primers were

designed to be on the 5' and 3' sides of the fusion junction of the desired sequence. The parent

plasmid DNA for the 5' sequence to be fused was chosen to be the recipient vector DNA, due to

the availability of convenient unique restriction sites. Therefore, the 3' outside primer was

designed to include a restriction site corresponding to a site available in the vector DNA. First

and second step PCRs were performed exactly as described above for two-step mutagenic PCR,

except the semi-overlapping primers (primers overlapping the fusion junction) were used in

place of the mutagenic primers. Here, the second step PCR product represents a new synthetic

gene fusion of the two previously distinct DNA sequences. Purified second step PCR product

was digested with the appropriate restriction enzymes and cloned into the corresponding sites of

the recipient vector DNA.

Construct Verification and DNA Stock Preparation

Small scale plasmid DNA of potential clones was prepared using either the Eppendorf or

Qiagen Mini-Prep Spin kits. The correctness of all constructs was verified by sequencing

performed either at the DNA Sequencing Core Laboratory (ICBR, University of Florida) or by

the SeqWright commercial sequencing facility (SeqWright, Inc., Houston, TX). All primary









clones generated using site directed mutagenesis were subsequently recloned back into the parent

vector background by excision and transfer of a sequence verified restriction fragment containing

the desired mutation. Although XL-1 Blue competent cells (Stratagene) were utilized for some

sub-cloning applications, all final plasmid DNAs were transformed into MAX Efficiency StBL2

competent cells (Invitrogen) for preparation of glycerol stocks as well as for large scale plasmid

DNA preparation. For long term storage, 50% glycerol stocks of overnight liquid cultures were

maintained at -800C and were re-struck on LB+ampicillin agar plates as needed. For large scale

plasmid DNA preparations, a single colony from a newly struck LB+ampicillin plate was

inoculated into 250 mL of LB broth containing 50 mg/mL ampicillin. These inoculated broth

cultures were grown overnight at 370C with shaking, and bacterial pellets were isolated by

centrifugation at 5,000 x g for 10 min. Plasmid DNA was subsequently isolated using the

Qiagen Midi-Prep kit. All plasmid DNA stocks were standardized to 0.5 gg/iL and stored in TE

[10 mM TrisHCl (pH=8), 1 mM EDTA] at -200C.

cDNA Clones Used in These Studies

Poliovirus Clones Used in These Studies

A previously described cDNA clone of the Mahoney strain of type I poliovirus, designated

pT7-PV1(A)so, was used as the parent clone for all poliovirus based constructs used in all studies

herein (26). (i) pPV1AGUA3 is a previously described construct which generates an RNA

transcript [PV1AGUA3 RNA] with a 5-nt deletion in the 3' NTR, known to inhibit

negative-strand synthesis without affecting translation (26). (ii) pP23 is a previously described

construct with a deletion of the P1 capsid coding region (104). RNA transcripts of this construct

[P23 RNA] express all essential replication proteins from the P2 and P3 regions of the viral

genome. These transcripts function as an RNA replicon, allowing for negative-strand, but not

positive-strand synthesis. (iii) pRzP23 was generated from pP23 by inserting a hammerhead









ribozyme (Rz) downstream of the T7 promoter. Following transcription, this ribozyme removes

itself from the 5' end of the transcript, yielding RNA transcripts with authentic poliovirus 5' ends

(25, 141). These authentic ended transcripts function as replicons, capable of both positive- and

negative-strand synthesis. (iv) pP23-5'CL(C24A) and pRzP23-5'CL(C24A) were engineered

using site-directed mutagenesis. Transcripts of these constructs contain the mutant 5'CLC24A, but

translate all viral replication proteins. (vi) pP23-5'CL(MS2) was generated using site-directed

mutagenesis, replacing nts 12-32 in pP23 (stem-loop b of 5'CL) with the cDNA for a 19 nt

stem-loop from the MS2 bacteriophage genome sequence [ACATGAGGATTACCCATGT]

(114). Proper folding of the resulting mutant 5'CLMS2 was verified using the Mfold RNA

structure prediction program (220, 221). (vii) pF3 is a previously described construct in which

the P1 and P2 coding regions were deleted, and a frameshift mutation was engineered near the

beginning of the P3 coding region (183). Transcripts of this construct [F3 RNA] initiate

translation at the 3A start codon but prematurely terminate translation. (viii) pF3-5'CL(C24A)

was generated from pF3 using site-directed mutagenesis and transcripts of this construct contain

the mutant 5'CLC24A. (ix) pFS23 is derived from pP23 via the deletion of nucleotides 775-779

by restriction digest, blunting with the Klenow fragment of T4 DNA polymerase, and re-ligation.

This deletion generated a reading frame shift causing transcripts of this construct [FS23] to

initiate translation at the 2A start codon but prematurely terminate after the synthesis of a 65

amino acid peptide. (x) pP1-3D* is derived from pT7-PV1(A)so via insertion of a Stul restriction

site at position 3364 by site-directed mutagenesis and subsequent removal of the Stul-Drall

fragment by restriction digest, blunting by T4 DNA polymerase, and re-ligation. The net effect

of this process is an in-frame deletion of nucleotides 3365-6082, spanning the extreme 3' end of

the P1 coding region through a 5' portion of the 3D coding region. Transcripts of this construct









[P1-3D* RNA] express a non-functional protein consisting of the majority of the P1 precursor

(amino acids 1-874) fused to a large carboxy-terminal portion of 3D (amino acids 13-460).

(xi) pFS 1-3D* is derived from pPl-3D* via the deletion of nucleotides 1119-1122 by restriction

digest, blunting with the Klenow fragment of T4 DNA polymerase, and re-ligation. This

deletion generated a reading frame shift causing transcripts of this construct [FS1-3D* RNA] to

initiate translation at the VP4 start codon but prematurely terminate after the synthesis of a 133

amino acid peptide. (xii) pP23-2ASTOP is derived from pP23 via insertion of two stop codons

following the terminal Gln codon of the 2A coding sequence by site-directed mutagenesis.

Transcripts of this construct [P23-2ASTOP RNA] retain the translation initiation context and all

other RNA sequences of P23 RNA but translation terminates at the precise carboxy-terminus of

2A. (xii) pP23-2BSTOP is derived from pP23 via insertion of two stop codons following the

terminal Gln codon of the 2B coding sequence by site-directed mutagenesis. Transcripts of this

construct [P23-2BSTOP RNA] retain the translation initiation context and all other RNA

sequences of P23 RNA but translation terminates at the precise carboxy-terminus of 2B.

(xiv) pP23-2CSTOP is derived from pP23 via insertion of two stop codons following the terminal

Gln codon of the 2C coding sequence by site-directed mutagenesis. Transcripts of this construct

[P23-2CSTOP RNA] retain the translation initiation context and all other RNA sequences of P23

RNA but translation terminates at the precise carboxy-terminus of 2C. (xv) pP23-3ASTOP is

derived from pP23 via insertion of two stop codons following the terminal Gln codon of the 3A

coding sequence by site-directed mutagenesis. Transcripts of this construct [P23-3ASTOP RNA]

retain the translation initiation context and all other RNA sequences of P23 RNA but translation

terminates at the precise carboxy-terminus of 3A. (xvi) pP23-3BSTOP is derived from pP23 via

insertion of two stop codons following the terminal Gln codon of the 3B coding sequence by









site-directed mutagenesis. Transcripts of this construct [P23-3BSTOP RNA] retain the translation

initiation context and all other RNA sequences of P23 RNA but translation terminates at the

precise carboxy-terminus of 3B. (xvii) pP23-3CSTOP is derived from pP23 via insertion of two

stop codons following the terminal Gln codon of the 3C coding sequence by site-directed

mutagenesis. Transcripts of this construct [P23-3CSTOP RNA] retain the translation initiation

context and all other RNA sequences of P23 RNA but translation terminates at the precise

carboxy-terminus of 3C. (xviii) pPVlp50 is derived from pT7-PV1(A)so via an in-frame

deletion of nucleotides 867-6011 by digestion with BstBI and re-ligation of the vector fragment.

Transcripts of this construct [PVlp50 RNA] utilize the authentic translational start and stop

contexts and express a non-functional fusion protein (p50) between a short VP4 peptide (amino

acids 1-41) and the majority of 3D (amino acids 9-460). (xix) p2BC-P3 is a previously described

construct which contains the coding region for the 2BC-P3 precursor protein (104). Transcripts

of this construct [2BC-P3 RNA] express the 2BC-P3 precursor protein. (xx) p2C-P3 is derived

from two previously described constructs p2C and pP23 via removal of the BamHI fragment

from p2C and subsequent ligation of that fragment into the corresponding sites of pP23 (104).

Transcripts of this construct [2C-P3 RNA] express a 2C-P3 precursor protein. (xxi) pP3 is a

previously described construct which contains the coding region for the entire P3 polyprotein

precursor (104). Transcripts of this construct [P3 RNA] express the P3 polyprotein precursor.

(xxii) p3BCD is derived from pP3 via site directed mutagenesis and subsequent removal of the

3A coding region. Briefly, a Smal restriction site, Kozak's consensus sequence and AUG codon

were introduced into pP3 immediately 5' of the initiating Gly codon of 3B. This mutant pP3

now contained tandem Smal sites, one immediately upstream of the P3 start codon, and one

immediately prior to the newly introduced 3B start codon. This subclone was digested with









Small, removing the 3A coding region, and subsequently re-ligated to form p3BCD. Transcripts

of this construct [3BCD RNA] express the 3BCD precursor protein. (xxiii) p3CD is derived by

an in-frame insertion of the 3C-3D(nt 1-247) protein coding sequence into the MscI site of a

previously described vector, pDJB2 (104). This vector retains the 5' and 3'NTR/poly(A) tail of

full-length cDNA clone pT7PV1(A)so, as well as a portion of 3D coding region, and contains the

Kozak's consensus translational start site. Transcripts of this construct [3CD RNA] express

proteolytically active 3CDpro precursor protein. (xxiv) p3D is derived by an in-frame insertion of

the 3D(nt 1-247) protein coding sequence into the MscI site of pDJB2, as described above.

Transcripts of this construct [3D RNA] express active 3DP"o alone. (xxv) To generate a series of

PV polyprotein expression RNAs that would act as partial helper RNAs, the AvrlI-Mlul fragment

from pPV1AGUA3 was transferred into the corresponding sites of p3D, p3CD, pP3, and

p2BC-P3, generating p3DAGUA3, p3CDAGUA3, pP3AGUA3, and p2BC-P3AGUA3,

respectively. Each of these generates transcripts which express the indicated portion of the PV

polyprotein but are defective for RNA replication. (xxvi) p3CD[3D-G327M]AGUA3 and

pP23 [3D-G327M] were created by transferring the BstBI-AvrlI fragment (containing the G327M

mutation) to p3CDAGUA3 or pP23, from pT7-PV1(A)so[3D-G327M] which had been generated

previously in our laboratory. Transcripts of these constructs [3CD(G327M)AGUA3 RNA or

P23[3D-G327M] RNA] express 3CD which processes and binds RNA normally, however, the

G327M mutation has disrupted the essential YGDD catalytic RNA polymerase motif, abolishing

polymerase activity (98). (xxvii) p3CD[PM]AGUA3 was created by mutagenic two-step PCR,

using p3CDAGUA3 as a template. This mutant combines two previously described processing

site mutations [T181K, Q182D] with two additional mutations [S183G, Q184N] designed to

completely abrogate 3C-3D processing (12, 37, 88). Transcripts of this construct









[3CD(PM)AGUA3 RNA] express 3CD which retains RNA binding and protease activity, but is

unable to be processed into 3C and active 3DP1l. (xxviii) pP23[3CD(PM)] was created by

transferring the BgllI-BstBI restriction fragment from p3CD[PM]AGUA3 into the corresponding

sites of pP23. (xxix) p3CD[3C-R84S]AGUA3 was created by mutagenic two-step PCR, using

p3CDAGUA3 as a template. This mutation was previously identified to inhibit the RNA binding

ability of 3Cpro (36). Transcripts of this construct [3CD(R84S)AGUA3 RNA] express a 3CD

with impaired RNA binding abilities, but retains the ability to release a fully wild-type 3DP1.

(xxx) pP23[3C-R84S] was created by transferring the BgllI-BstBI restriction fragment from

p3CD[PM]AGUA3 into the corresponding sites of pP23. (xxxi) p3CD[3C-K12N/R13N]AGUA3

and pP23[3C-K12N/R13N] were created by mutagenic two-step PCR, using p3CDAGUA3 or

p23 as templates, respectively. These mutations were previously identified to inhibit the RNA

binding ability of 3Cpro (36).

Poliovirus-Based Protein Expression Clones Used in These Studies

As described above, pDJB2 containing the AGUA3 mutation was used as a PV expression

vector to direct translation of a downstream reading frame (104). As before, all protein

expression clones generate AGUA3 RNA transcripts, which prevents the transcripts from

functioning as RNA replicons (26). This vector can be digested with MscI which cuts directly

downstream from the IRES, prior to any initiating AUG codons. PCR products containing the

Kozak's consensus translation initiation sequence and the coding region of a gene of interest can

then be ligated into this vector, effectively replacing the PV coding sequence with that of the

desired protein. (i) cDNA clones containing the PCBP1 and PCBP2 coding sequence were

generously provided by Dr. Raul Andino (77). The PCBP1 and PCBP2 coding sequences were

PCR amplified from plasmid DNA and cloned into the MscI site of pDJB2AGUA3 as described

above, generating the PCBP1 and PCBP2 expression constructs pPCBP1AGUA3 and









pPCBP2AGUA3. (ii) The MS2 coding sequence (137) was synthesized by GeneArt, optimizing

codon usage for both mammalian and bacterial expression. The synthetic DNA contained a 5'

Small site as well as consensus Kozak's sequence upstream of the initiating AUG. For

expression and cloning purposes, the MS2 coding sequence was tailed with a 3' RGSH linker

(112) and the 5' nucleotides of the PCBP2 coding sequence through the preexisting Alel site.

This synthetic MS2 DNA was digested and cloned into the Smal and Alel sites of the

pPCBP2AGUA3 expression construct, generating an in-frame fusion of MS2 and PCBP2

(pMS2-PCBP2AGUA3). Using two-step PCR, a second MS2 coding region was fused, in-frame,

upstream of the original MS2-PCBP2 sequence, generating the p(MS2)2PCBP2AGUA3

expression construct, in which the two MS2 sequences were joined with a GAPGIHPGM peptide

linker, described by Hook et al. (96). (iii) The (MS2)2 expression construct [p(MS2)2AGUA3]

was generated by introducing two stop codons in the (MS2)2PCBP2AGUA3 plasmid, following

the RGSH linker sequence, using site-directed mutagenesis. The PCBP2 coding sequence was

then removed from this construct by restriction digest and re-ligation. (iv) The (MS2)2PCBP1

expression construct [p(MS2)2PCBP1 AGUA3] was generated by semi-overlapping two-step PCR

using p(MS2)2AGUA3 and pPCBP1AGUA3 as templates. The resultant fused PCR product was

ligated into the XmaI and Xhol sites of p(MS2)2AGUA3. (v) The PCBP2-KL and

(MS2)2PCBP2-KL expression constructs [pPCBP2-KLAGUA3 and p(MS2)2PCBP2-KLAGUA3]

were generated by deleting the cDNA corresponding to exon 8a in each parent clone using

two-step mutagenic PCR. (vi) The (MS2)2KH1 [Region] expression construct

[p(MS2)2KH1 [Region]AGUA3] was generated by PCR amplification of the coding sequence for

amino acids 1-91 of PCBP2 using a 5' phosphorylated (5'P04) primer and a 3' primer containing

an Xhol site. This product was digested with Xhol and ligated into the Alel and Xhol sites of









p(MS2)2PCBP2AGUA3, essentially replacing the PCBP2 coding sequence downstream of the

RGSH linker. (vii) The (MS2)2KH2[Region] expression construct

[p(MS2)2KH2[Region]AGUA3] was generated by PCR amplification of the coding sequence for

amino acids 90-233 of PCBP2 and subsequent cloning into the XhoI and Alel sites of

p(MS2)2PCBP2AGUA3, as described above. (viii) The (MS2)2KH2[Region] expression

construct [p(MS2)2KH3[Region]AGUA3] was generated by PCR amplification of the coding

sequence for amino acids 234-364 of PCBP2 and subsequent cloning into the Xhol and Alel sites

of p(MS2)2PCBP2AGUA3, as described above. (ix) The (MS2)2KH1/2[Region] expression

construct [p(MS2)2KH1/2[Region]AGUA3] was generated by PCR amplification of the coding

sequence for amino acids 1-233of PCBP2 and subsequent cloning into the Xhol and Alel sites of

p(MS2)2PCBP2AGUA3, as described above. (x) The (MS2)2KH3 [Domain] expression construct

[p(MS2)2KH3[Domain]AGUA3] was generated by PCR amplification of the coding sequence for

amino acids 269-357 of PCBP2 and subsequent cloning into the Xhol and Alel sites of

p(MS2)2PCBP2AGUA3, as described above. (xi) The (MS2)2KH3-Apl expression construct

[p(MS2)2KH3-AplAGUA3] was generated by PCR amplification of the coding sequence for

amino acids 280-357 of PCBP2 and subsequent cloning into the XhoI and Alel sites of

p(MS2)2PCBP2AGUA3, as described above. Transcripts of this construct express an (MS2)2

fusion to the KH3 domain with the first P-strand of the domain deleted. (xii) The

(MS2)2KH3-Aa3 expression construct [p(MS2)2KH3-Aa3AGUA3] was generated by PCR

amplification of the coding sequence for amino acids 269-340 of PCBP2 and subsequent cloning

into the XhoI and Alel sites of p(MS2)2PCBP2AGUA3, as described above. Transcripts of this

construct express an (MS2)2 fusion to the KH3 domain with the last a-helix of the domain

deleted. (xiii) The (MS2)2KH1/2-AMD expression construct [p(MS2)2KH1/2-AMD] was









generated by deleting the coding sequence for amino acids 125-158 of PCBP2 from the context

of p(MS2)2KH1/2[Region] using two-step mutagenic PCR. Transcripts of this construct express

an (MS2)2 fusion to the amino-terminal KH1/2 fragment of PCBP2 with a deletion in the

multimerization domain of KH2.

To obtain cDNAs for the remaining PCBP family members (PCBP3, PCBP4, PCBP4A,

and hnRNP-K), total cellular mRNA was obtained from 106 HeLa S3 suspension cells using

TRIzol Reagent (Invitrogen) and subsequent ethanol precipitation. Using this purified cellular

mRNA, 1st-strand cDNA was generated using SuperScript III Reverse Transcriptase (Invitrogen)

with either Oligo(dT)20 or DNA primers complimentary to the 3'NTR of the gene of interest.

Digestion of the parent mRNA was achieved using RNase H (Invitrogen) and 2nd-strand cDNA

was generated using PfuUltra Fusion Polymerase (Stratagene) with primers complimentary to the

5'NTR of the gene of interest. This newly synthesized cDNA was further amplified using

PfuUltra Fusion Polymerase (Stratagene) and 5'PO4 versions of the same 5' and 3' primers as

were used for 1st and 2nd-strand cDNA synthesis. These specific PCR products were subcloned

into the EcoRV site of pLITMUS39 (New England Biolabs) for blue/white screening. Following

restriction enzyme screening, each coding sequence was PCR amplified from a correct subclone

using 5' and 3' P04 primers specific to the precise gene start and stop. In addition, the 5' primer

contained the Kozak's consensus sequence immediately prior to the initiating ATG, and the 3'

primer contained an additional stop codon to prevent any potential translational read-through. In

all cases, the PCR products were ligated into the MscI site of pDJB2AGUA3, generating the

expression constructs pPCBP3AGUA3, pPCBP4AGUA3, pPCBP4AAGUA3, and

pHnRNP-KAGUA3. The coding sequence for each of these PCBP family members was further

fused to the (MS2)2 coding sequence by semi-overlapping two-step PCR and ligation into the









Xmal and Xhol sites of p(MS2)2AGUA3. This resulted in the generation of the expression

constructs p(MS2)2PCBP3AGUA3, p(MS2)2PCBP4AGUA3, p(MS2)2PCBP4AAGUA3, and

p(MS2)2hnRNP-KAGUA3.

Bacterial Protein Expression Clones Used in These Studies

To inducibly express proteins of interest, the pET16b plasmid DNA was obtained from

Novagen. Although this vector contains sequences encoding a previously inserted

amino-terminal polyhistidine tag, these sequences were removed as a result of the cloning

process. This would then result in the expression of fully wild-type, untagged protein following

transfection and induction in expression cells. (i) To generate the bacterial expression constructs

for PCBP1, PCBP2, PCBP2-KL, and PCBP3, the coding sequence for each protein was removed

from the corresponding poliovirus based expression clone (described above) using Ncol and

Xhol. The resultant fragment was ligated into the corresponding sites of similarly digested

pET16b plasmid DNA, generating pET16-PCBP1, pET-PCBP2, pET16-PCBP2KL, and

pET16-PCBP3. (ii) To generate the bacterial expression constructs for PCBP4 and PCBP4A,

each coding sequence was amplified from each of the poliovirus expression constructs using a 5'

primer containing a BspHI restriction site and a 3' primer containing an Xhol restriction site,

since BspHI generates an NcoI-compatible overhang. Each PCR product was digested with

BspHI and Xhol, and subsequently ligated into an Ncol/Xhol digested pET16b vector DNA,

generating pET16-PCBP4 and pET16-PCBP4A. (iii) To generate the bacterial expression

construct for hnRNP-K, pET16b was digested with Xhol, filled in with the Klenow fragment of

T4 DNA polymerase, and then further digested with Ncol. The entire coding sequence for

hnRNP-K was removed from the poliovirus expression construct by digestion with Ncol and

Pmel (blunt cut). This fragment was subsequently ligated into the corresponding sites of the

Ncol/Xhol-blunt pET16b vector DNA, generating pET16-hnRNPK









RNA Transcript Preparation and Purification

Prior to in vitro transcription, the run-off transcription template was prepared by digesting

the desired plasmid DNA with Mlu. Digestion with this enzyme resulted in linearization of the

circular plasmid DNA via a single cut immediately following the poliovirus 3'NTR/poly(A) tail.

Restriction digest reactions were phenol: chloroform extracted three times, chloroform extracted

three times, and subsequently ethanol precipitated. Ethanol precipitated Mlu cut template DNA

was resuspended in TE, standardized to 0.5 tgg/tL, and stored at -200C.

Standard Transcription

Standard transcription conditions were used for generating all non-capped, non-ribozyme

transcript RNAs. In these conditions, transcription reactions contained 1X transcription buffer

[40 mM TrisHCl (pH=8), 6 mM MgC12, 2 mM spermidine], 10 mM DTT, 0.4 U/ptL RNasin

(Promega), 1000 tM of each NTP (ATP, CTP, GTP, UTP), and 15 ng/[tL linearized template.

Bacterially expressed recombinant T7 polymerase was purified by B. Joan Morasco, and

approximately 1 [tL of this purified T7 polymerase was used per 100 [tL transcription reaction.

Reactions were incubated at 370C for 2 h and were stopped by the addition of 2.5 volumes of

0.5% SDS buffer [10 mM TrisHCl (pH=7.5), 100 mM NaC1, 1 mM EDTA, 0.5% sodium

dodecyl sulfate].

For purification purposes, RNA transcripts were phenol:chloroform extracted three times,

chloroform extracted three times, and subsequently precipitated by the addition of three volumes

of 100% ethanol and incubation overnight at -200C. Precipitated RNAs were further purified by

desalting over Sephadex G-50 (GE Healthcare) gel filtration resin (0.5 x 15 cm column). Peak

fractions containing RNA were identified and quantitated spectrophotometrically. All fractions

containing significant quantities of RNA were pooled, aliquotted (10-20 tg/aliquot), and ethanol









precipitated. These purified, desalted transcript RNAs were stored, in ethanol, at -200C and were

only precipitated immediately prior to their use in translation/replication experiments.

Ribozyme Optimized Transcription

For RNA transcripts containing the Rz sequence, transcription conditions were altered to

optimize Rz cleavage (141). These conditions are identical to those provided above for standard

transcription, with the exception of the NTP concentrations. Here, each NTP (ATP, CTP, GTP,

UTP) was included in the transcription reaction at 500 riM. It was determined experimentally

that these conditions resulted in >95% ribozyme cleavage efficiency. Ribozyme optimized

transcription reactions were stopped, extracted, desalted, and stored in the same manner as

described above for standard transcription reactions.

5' Capping Transcription

To synthesize RNAs with a m7G cap analog, transcription conditions were slightly altered

to optimize the capping reaction. These conditions are identical to those provided above for

standard transcription, with the exception that the GTP concentration was lowered to 200 riM,

and 800 [iM of m7G[5']ppp[5']G cap analog (Epicentre) was added to the transcription reaction

mixture (26). Under these conditions, -80% of the transcript RNAs contain a 5' 7mG cap.

Capping transcription reactions were stopped, extracted, desalted, and stored in the same manner

as described above for standard transcription reactions.

HeLa Extract Preparation

HeLa S3 cells were adapted to liquid suspension culture and were maintained in Joklik's

modified Eagle medium supplemented with 5% bovine calf serum (Hyclone) and 2%

FetalClone II (Hyclone). Cells were passage as needed to maintain a cell density of less than

5 x 105 cells/mL. In cases where cells were being grown for the purposes of an S10 preparation,

HeLa cells were pelleted and resuspended in fresh 18-24 h prior to their use in the S10









procedure. The following procedures were originally developed in our laboratory by Barton et

al, and are described in more detail in previous publications (21, 22, 24). Any deviation from

these published procedures has been noted, where applicable.

S10 Preparation

HeLa cell density was determined by hemocytometer count, and approximately 109 HeLa

suspension cells were pelleted by low speed centrifugation. This cell pellet was washed

sequentially with 2 L of isotonic buffer [35 mM HEPES-KOH (pH=7.4), 146 mM NaC1, 5 mM

dextrose], and was resuspended over 10 min on ice in 1.5 volumes of hypotonic buffer [20 mM

HEPES-KOH (pH=7.4), 10 mM KC1, 1.5 mM Mg(CH3CO2)2, 1 mM DTT] with gentle

vortexing. Resuspended swollen cells were transferred to a glass dounce homogenizer and were

dounced on ice using a type 'A' or tight pestle. Cell integrity was monitored by removing a

small aliquot at various times during douncing and visually assessing percent lysis by light

microscopy. Optimal lysis was defined as approximately 80% lysis with visibly intact nuclei and

typically required 20-25 strokes of the dounce. The final volume of this mixture was determined

and 1/9 volume of 10X S10 buffer [200 mM HEPES-KOH (pH=7.4), 1.2 M K(CH3CO2), 40 mM

Mg(CH3CO2)2, 50 mM DTT] was added to make the final solution IX. Following this addition,

unlysed cells, nuclei, and other dense debris were removed by low speed centrifugation. The

resultant semi-cleared supernatant was subsequently transferred to a siliconized corex tube and

centrifuged at 12,000 x g for 15 min at 40C. The supernatant from this centrifugation was treated

with micrococcal nuclease (5 [g/mL, 200C, 15 min) in the presence of CaC12 (1 mM) to degrade

all endogenous cytoplasmic cellular mRNAs. After micrococcal nuclease treatment, the

nuclease was inactivated by addition of EGTA (2 mM) to chelate the essential calcium. Any

additional insoluble debris was the removed by a second 15 min centrifugation at 12,000 x g.

The supernatant from this spin was divided into single use aliquots and stored at -800C. This









supernatant is defined as HeLa S10 for the purposes of all experiments performed in the studies

herein.

IF Preparation

The initial procedure for IF preparation is exactly the same as described above for S10

preparation with three significant changes: 1)Washed cell pellets were resuspended in 2 volumes

of hypotonic buffer, rather than 1.5 volumes. 2)Swollen cells were dounced to 90-100% lysis,

rather than 80%, however minimal disruption of nuclei is still ideal. 3)The supernatant from the

first 12,000 x g centrifugation is not treated with micrococcal nuclease.

The supernatant from the first 12,000 x g centrifugation described above is transferred to

an ultracentrifuge tube and centrifuged at 330,000 x g for 60 min at 40C. The pellet from this

spin contains the cytoplasmic ribosomes and ribosome associated protein components, as well as

the smooth and rough endoplasmic reticulum, microsomes, exosomes, and other lipid related

structures. The supernatant was removed and the ribosome containing pellet was resuspended in

1.5 mL of hypotonic buffer. Resuspension of this pellet was facilitated by the use of a magnetic

micro stir bar and stir plate at 40C, and typically required 30 min stir time. To standardize

protein preparations, a 2 [tL aliquot was removed, diluted 1:250, and the absorbance at 260 nm

was obtained. This roughly reflects the concentration of ribosomal RNA in the preparation, and

readings in the range of 0.7-0.9 (175-225 A260 units undiluted) reflect the optimal concentration

range. Preparations that exceed this range were diluted appropriately with hypotonic buffer until

the desired absorbance is reached. Once an optimal A260 reading was attained, the total volume

was measured and 1/7 volume of 4 M KC1 was added. This addition raised the final KCl

concentration to 0.5 M, disrupting the ionic and electrostatic interactions necessary for initiation

factor association with the larger ribosomal complex. To allow this dissociation to proceed, the

mixture was incubated for 15 min at 40C with stirring. The mixture was subsequently









centrifuged again at 330,000 x g for 60 min at 40C. The resultant supernatant was removed and

dialyzed against IF buffer [20 mM HEPES-KOH (pH=7.4), 120 mM K(CH3CO2), 5 mM

Mg(CH3CO2)2, 5 mM DTT] for 2 h at 40C. The dialyzed supernatant was divided into single use

aliquots and stored at -800C. This dialyzed supernatant is defined as HeLa IF for the purposes of

all experiments performed in the studies herein.

HeLa S10 Translation-RNA Replication Reactions

For all experiments, extract preparations are thawed on ice immediately prior to their use,

and aliquots are never reused once thawed. Additionally, transcript RNAs are stored in ethanol

and are precipitated, washed, resuspended and quantitated immediately prior to their use.

Transcript RNAs, once precipitated, were never reprecipitated and reused for later experiments.

In general, all reaction components are thawed and stored on ice during experimental setup.

Both translation and replication reactions utilized single use aliquots of a 10X nucleotide

reaction mix [155 mM HEPES-KOH (pH=7.4), 600 mM K(CH3COOH), 300 mM creatine

phosphate, 4 mg/mL creatine phosphokinase, 10 mM ATP, 2.5 mM GTP, 2.5 mM UTP]. This

reaction mix includes optimal buffers and salts for PV translation and an ATP regenerating

system, but excludes CTP. This omission allows for later use of [32P]CTP to radiolabel RNA

replication products.

RNA Programming and Translation

HeLa S10 translation reactions were prepared by combining 50% (by volume) HeLa S10

extract, 20% (by volume) HeLa IF, 10% (by volume) 10X nucleotide reaction mix, 2 mM

guanidine hydrochloride (GuHC1), template RNA, and sterile/RNase-free water. Purified

template RNA was precipitated, resuspended in sterile/RNase-free water, and quantitated

spectrophotometrically. Unless otherwise specified in an individual experimental methodology,

4 pmol of purified template RNA was used to program the HeLa S10 translation reactions. In









cases where multiple RNAs were required (e.g. trans-replication experiments), 4 pmol total

RNA was used. When RNA programmed translation reactions were assembled, a 10 [tL aliquot

was removed, to which 11 I Ci (1 pL) of L-[35S]-methionine (1,000 Ci/mmole; PerkinElmer) was

added for metabolic labeling of newly synthesized proteins. Both the HeLa S10 translation

reactions and the [35S]-methionine labeling side reaction were incubated at 340C for 3-4 h.

Following incubation, the HeLa S10 translation reactions were centrifuged to isolate

pre-initiation replication complexes (PIRCs), which is described below. Additionally, 5 |iL of

the [35S]-methionine labeling side reaction was added to 45 tiL of 1X Laemmli sample buffer

(LSB; 112.5 mM TrisHCl (pH=6.8), 2% sodium dodecyl sulfate, 20% glycerol, 0.5%

P-mercaptoethanol, 0.02% bromophenol blue). This mixture was stored at -200C prior to

analysis by SDS-PAGE.

PIRC Isolation and RNA Replication

Membrane associated PV translation in the HeLa S10 translation reactions resulted in the

formation of replication complexes. The inclusion of GuHCl in the translation reaction allowed

these complexes to form, however initiation of negative-strand synthesis was blocked. These

pre-fire complexes have been defined as pre-initiation replication complexes (PIRCs). PIRCs

can be obtained by centrifugation and isolation of the membrane pellet from HeLa S10

translation reactions. To do so, HeLa S10 translation reactions were centrifuged at 20,000 x g

for 15 min at 40C. Supernatants were carefully removed so as not to disturb the membrane

pellet. PIRC pellets were gently resuspended in replication buffer, which contained 50% (by

volume) S10 buffer (40 mM HEPES-KOH (pH=7.4), 120 mM K(CH3CO2), 5.5 mM

Mg(CH3CO2)2, 6 mM DTT, 10 mM KC1], 10% (by volume) 10X nucleotide reaction mix,

0.1 mg/mL puromycin, 5 tM CTP, and 30 iCi [a-32P]CTP (800 Ci/mmol; PerkinElmer).

Resuspension in a GuHCl-free buffer washed out the residual inhibitory GuHCl from the PIRCs,









and replication was allowed to proceed by incubation at 370C for 1 h. Following incubation,

RNA replication reactions were stopped with 6 volumes of 0.5% SDS buffer, and were treated

with 20 tg of proteinase K for 15 min at 370C. Digested RNA replication reactions were

subsequently extracted three times with phenol:chloroform, extracted three times with

chloroform, and precipitated by the addition of 3 volumes of 100% ethanol. Extracted product

RNAs were stored in ethanol at -200C for at least 12 h, or until analyzed by gel electrophoresis.

Analysis of Protein Synthesis by SDS-PAGE

Protein synthesis was analyzed by 9-18% gradient sodium dodecyl sufate polyacrylamide

gel electrophoresis (SDS-PAGE). To do this, a vertical 0.75 mm 9-18% gradient resolving gel

was cast using a gradient maker, and a 4% stacking gel was cast above the gradient gel. The

ratio of acrylamide to bis-acrylamide used was 29:1, and the standard Tris-glycine discontinuous

buffer system was also used. Samples were electrophoresed at constant current until the

bromophenol blue dye front had exited the bottom of the resolving gel. Completed gels were

fixed in 40% methanol/10% acetic acid for at least 15 min and rinsed with deionized water.

Fixed gels were then impregnated with Amplify Fluorographic Reagent (GE Healthcare) by

soaking for 10 min. Residual Amplify was rinsed away using deionized water and gels were

transferred to chromatography paper for drying. Autoradiography was performed by exposure of

dried gels to Kodak X-omat Blue XB-1 scientific imaging film at either -200C or -800C. Where

applicable, quantitation of protein products was performed by phosphorimager using

ImageQuant software (Molecular Dynamics).

Analysis of RNA Replication by Denaturing CH3HgOH Gel Electrophoresis

Due to the uniquely elevated stability of an extended RNA-RNA duplex, complete

denaturation of product RNA, particularly negative-strand RNA in a replicative form duplex,

requires a powerful denaturant. For this reason, methyl mercury hydroxide (CH3HgOH) agarose









gel electrophoresis was utilized to resolve and visualize RNA products from HeLa S10

translation-RNA replication reactions. Purified product RNA was recovered from ethanol

precipitation by centrifugation, washing, and resuspension in 15 itL of sterile/RNase-free water.

An equal volume of CH3HgOH sample buffer [50 mM H3B03/5 mM Na2B207 (pH=8.2), 10 mM

Na2SO4, 1 mM EDTA, 25% glycerol, 0.05% bromophenol blue, 50 mM CH3HgOH] was then

added to the resuspended replication product, and allowed to denature for 5-15 min at room

temperature. Denatured RNA products were resolved on a vertical 1% Seakem LE agarose gel

which contained 5 mM CH3HgOH. Electrophoresis was performed at 70 mA constant current in

IX CH3HgOH running buffer [50 mM H3BO3/5 mM Na2B207 (pH=8.2), 10 mM Na2SO4, 1 mM

EDTA]. For the first hour of electrophoresis, the buffer in the upper and lower buffer chambers

were recirculated using a peristaltic pump to avoid depletion of buffering capacity.

Electrophoresis was halted when the bromophenol blue dye front reached 1-2 cm from the

bottom of the gel (typically 2.5 h total time). Gels were stained with 1.0 mg/mL ethidium

bromide in 0.5 M NH4(CH3COOH) for 10 min and visualized on a UV transilluminator to

ascertain equal loading/recovery. Gels were subsequently transferred to chromatography paper

for drying. Autoradiography was performed by exposure of dried gels to Kodak X-omat Blue

XB-1 scientific imaging film at -800C using a Biomax intensifying screen. Quantitation of RNA

products was performed by phosphorimager using ImageQuant software (Molecular Dynamics).

Bacterial Protein Expression

Bacterial protein expression plasmids were maintained as DNA stocks in TE at -200C and

were only transformed into expression cells immediately prior to protein expression. Plasmid

DNA was transformed into BL21(DE3) pLysS competent cells (Novagen) and a single colony

was used to inoculate a 5.0 mL LB broth culture containing 50 tg/mL ampicillin and 34 tg/mL

chloramphenicol. This culture was incubated overnight at 370C with shaking. A fresh 50 mL









LB broth culture without antibiotic was then inoculated with 50 [L of the overnight culture and

incubated at 250C with shaking. Protein expression was induced for 2 h with 1 mM IPTG when

the culture reached an OD600 of -0.5. Cleared protein extracts were prepared as described in

Andino et al (12). Briefly, cells were harvested, weighed, washed once with phosphate buffered

saline, and resuspended using 5.0 mL/g wet weight in lysis buffer (10 mM HEPES-KOH

(pH=7.9), 20 mM KC1, 25 mM EDTA, 5 mM DTT, 1% Triton X-100). The resuspended cell

pellet was frozen and thawed, sonicated for 30 s, and centrifuged at 150,000 x g for 15 min. The

supernatant from this centrifugation was supplemented with glycerol to a final concentration of

20%, aliquotted and stored at -800C.

Electrophoretic Mobility Shift Assays

Riboprobe Synthesis

DNA templates containing the cDNA for the PV 5'CL were digested with Hgal, which

cuts 30-nts past the 5'CL. Cut DNAs were purified by phenol:chloroform extraction and ethanol

precipitation. Radiolabeled 5'CL probes were made by T7 transcription of Hgal cut DNA

template as described above for ribozyme optimized transcription, with one exception. Rather

than 500 [M CTP, a combination of 115 [M non-labeled CTP and 4 Ci/iL [a-32P]CTP (400

Ci/mmol) was used. Probes were purified directly from the transcription reaction by passage

over NucAway Spin Columns (Ambion), followed by a single phenol:chloroform extraction,

single chloroform extraction, and ethanol precipitation. Riboprobes were stored in ethanol at

-200C until immediately prior to their use, and were only reprecipitated for a maximum of two

additional experiments. For quantitation of precipitated radiolabeled riboprobes, TCA

precipitation, filtering, and scintillation counting was performed. Calculations were

subsequently performed based on the specific activity of the [32P]CTP in the transcription

reaction and the number of C residues in the given riboprobe.









Binding Reactions and Gel Electrophoresis

Electrophoretic mobility shift assays (EMSA) were performed based on a modified

protocol described previously (13). Radiolabeled riboprobes were prepared as described above,

and protein was either obtained from HeLa S10 translation reactions or as recombinant protein

from clarified bacterial lysate. Binding reactions were performed by pre-incubating 1.5 tL of

HeLa S10 translation reaction mixture or 0.5-3 tL of bacterial extract in a 9 gL reaction,

containing binding buffer [5 mM HEPES-KOH (pH=7.9), 25 mM KC1, 2 mM MgC12, 3.8%

glycerol, 1.5 mM ATP, 20 mM DTT], 20 tg yeast tRNA, and 40 U RNasin (Promega) at 300C

for 10 min. To this preincubation mix, 20 fmol of 32P-labeled riboprobe was added to the

reaction. The final binding reaction was incubated at 300C for an additional 10 min prior to

addition of 2 [L loading buffer [0.1% bromophenol blue, 50% glycerol]. A 5% polyacrylamide

[40:1 acrylamide:bisacrylamide] native gel containing 5% glycerol was cast in 0.5X TBE buffer

[176 [M TrisHC1, 176 IM H3BO3, 2 mM EDTA]. Prior to loading, the gel was pre-run at 40C

for 30 min at 30 mA with constant current using 0.5X TBE as the running buffer.

Ribonucleoprotein complexes were resolved by electrophoresis at 40C at 220 V with constant

voltage. Electrophoresis was halted when the bromophenol blue dye front reached 3-4 cm from

the bottom of the gel. The final gel was transferred to chromatography paper, dried, and

visualized by autoradiography as described above.









CHAPTER 3
POLY(C) BINDING PROTEIN IS REQUIRED FOR EFFICIENT INITIATION OF
POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS

Introduction

The poly(C) binding proteins (PCBP; also called hnRNP E and aCP) represent a family of

poly(rC/dC) binding proteins which include hnRNP K and PCBPs 1-4 (109, 121, 128, 133). In

addition to their nucleic acid binding specificity, this protein family is characterized by the

presence and positioning of three highly homologous hnRNP K Homology domains(KH

domains) (80, 188). In the case of the PCBPs, the first and third KH domains contain the

primary nucleic acid binding activity, although the second domain may enhance binding affinity

and/or specificity (65, 67). It is thought that the structure of this domain is highly conserved,

regardless of surrounding sequence context, acting as an independent cassette which can be

evolutionarily tuned to a specific function. Although initially characterized as RNA binding

proteins involved in pre-mRNA metabolism, more recent work has described an increasingly

globalized set of essential cellular processes in which PCBPs participate. While there is some

degree of overlap in the sequences bound by the PCBPs, the use of alternate binding partners

together with modulation of binding specificity and affinity, results in an immense number of

potential regulatory targets and functions.

As yet, the most extensively studied family members are hnRNP K, PCBP1, and PCBP2.

Current work has firmly established the involvement of the PCBP protein family in mRNA

stabilization, transcriptional regulation, translational control, and apoptotic program activation

(reviewed by (129)). The mRNAs targeted by these proteins are diverse as well, including

a-globin, 15-lipoxygenase, collagen al, tyrosine hydroxylase, erythropoietin and androgen

receptor (49, 64, 154, 162, 191, 211, 217). In addition, more recent work identified over

150 mRNAs in a hematopoietic cell line that interact, in vivo, with PCBP2 alone (208). A









number of interacting proteins have also been identified, including AUF 1, HuR, SRp20 and

Poly(A) Binding Protein, as well as other members of the PCBP family (6, 28, 49, 77, 93, 108,

110).

Given the abundance and multi-functional nature of these proteins, it is not surprising that

multiple viruses, both RNA and DNA alike, have evolved to utilize these proteins during various

stages of their replication. The ORF57 protein of Kaposi's Sarcoma-associated herpesvirus

(KSHV) has been shown to interact with PCBP1, and this complex is capable of stimulating the

translation of specific cellular and viral genes (147). In the case of human papillomavirus

(HPV), PCBP1 interacts with one of the capsid protein mRNAs and down-regulates its

translation (60). Interestingly, recent work revealed markedly decreased levels of PCBP1 in

cervical epithelial cells transformed by HPV, and demonstrated a direct correlation between

cellular PCBP1 levels and progression to cervical cancer (169). Hepatitis C virus (HCV) binds

PCBP2 at both the 5' and 3' NTR of its genomic RNA (189, 200), however the role of these

binding events is yet to be understood.

Many members of the family Picornaviridae have also been shown to utilize the PCBPs

for their replication, including hepatitis A virus (HAV), human rhinovirus 14 (HRV-14),

coxackievirus B3 (CVB3) as well as poliovirus (PV). One functional commonality is the

requirement for PCBP2 in the cap-independent initiation of translation mediated by a type-I

IRES (38, 66). Interestingly, although HAV does not have a classic type I IRES, PCBP2 is still

utilized for translation initiation via interaction with an alternative sequence element in the 5'

NTR (83). Even the type-II IRES of Encephalomyocarditis Virus (EMCV), which does not

require PCBP, will still compete for PCBP binding (66). This suggests a more generalized

function for the PCBPs in picomaviral translation initiation.









Poliovirus (PV), in addition to the IRES, possesses a 5'-terminal cloverleaf (5'CL)

structure that is essential for RNA replication and is conserved among all members of the

Enterovirus genus (12, 13, 26, 93, 126, 196, 213, 219). The 5'CL is divided into four domains:

stem a and stem-loops b, c, and d (Figure 3-1A). Stem-loop dbinds viral protein 3CDPro, and

stem-loop b binds PCBP1 or PCBP2 (12, 13, 158). PCBP1/2 will bind to the 5'CL in the

absence of viral proteins, however the concomitant binding of 3CDpro results in a nearly 100-fold

increase in PCBP binding affinity (79). While both the first and third KH domains bind poly(C)

RNA with similar affinity, only the first KH domain of PCBP1/2 is required to bind to the 5'CL

(65, 185, 209). The binding of PCBP1/2 and 3CDpro to the 5' cloverleaf is believed to play an

important role in viral RNA replication (12, 13, 79, 213), and in RNA stability (144). In

addition, PV RNA replication is inhibited in Poly(C)-depleted HeLa S10 extracts, strongly

suggesting that PCBP binding to the 5' cloverleaf is required in one or more steps of the viral

RNA replication cycle (209).

In the current study, we investigated the role of the PCBP-5'CL RNP complex in PV RNA

replication. Herein, we present data that demonstrates that the binding of PCBP to the 5'CL is

required to form the replication complex used to initiate PV negative-strand RNA synthesis.

Furthermore, we describe the novel application of a protein-RNA tethering system in the

functional analysis of essential cellular protein involvement in virus replication. Using this

system, we were able to overcome the difficulties presented in performing experiments involving

RNAi, gene knockout, protein depletion or dominant negative inhibition of multi-functional,

essential cellular proteins, such as the PCBPs. Moreover, we demonstrate the ability of this

system to directly analyze and modify domains of a cellular protein, specifically as it pertains to

virus replication.









Results

A Mutation in Stem-loop b of the 5' Cloverleaf Inhibits Negative-strand Synthesis

To clarify the role of PCBP binding to the 5'CL in PV RNA replication, we used a

subgenomic PV RNA transcript [P23 RNA] which encodes all of the essential viral replication

proteins and forms functional RNA replication complexes in cell-free reactions. We compared

the replication of wild-type P23 RNA with the replication of the same RNA with a C24A

mutation in stem-loop b of the 5' CL [P23-5'CLC24A RNA] (Figure 3-1B). PV RNA replication

was assayed using preinitiation RNA replication complexes (PIRCs) isolated from HeLa S10

translation-replication reactions as described in Chapter 2. To assay for negative-strand

synthesis, we utilized P23 RNA transcripts which contain two 5' terminal non-viral G's that

inhibit positive-strand initiation (25, 141).

To first examine the ability of the 5'CLC24A to bind PCBP, we performed electrophoretic

mobility shift assays (EMSA) using radiolabeled 5'CL riboprobes containing either a wild-type

or C24A stem-loop b. The C24A mutation has been previously shown to inhibit the formation of

the essential 5' RNP complex (12, 77, 144, 158), and as expected, the PCBP-5'CL RNP complex

(complex I) was observed using 5'CLwT RNA probe but not on 5'CLC24A RNA probe. This

demonstrated that PCBP2 does not bind to 5'CLC24A RNA, as predicted. In RNA replication

reactions containing P23-5'CLC24A RNA, negative-strand synthesis was 10-20% of the amount

observed with wild-type P23 RNA (Figure 3-2B). Additional work from our lab has shown that

there is no defect in positive-strand synthesis of P23-5'CLC24A RNA, despite the decreased levels

of negative-strand synthesis (Sharma et al., unpublished results). Taken together, these

experiments demonstrated that P23-5'CLC24A RNA was defective for negative- but not

positive-strand synthesis









(MS2)2 Protein-RNA Tethering System

Tethered function assays have been used to study the activity of cellular proteins in mRNA

metabolism and regulation apart from their RNA binding affinity and specificity (reviewed in

57). One system that is used for this type of assay takes advantage of the high-affinity

interaction of the MS2 bacteriophage coat protein with its cognate RNA stem-loop structure (47,

58). The MS2 tethered function system requires the generation of an in-frame fusion of the MS2

coat protein with the protein of interest. At the same time, the native protein binding site in the

target RNA is replaced with the MS2 RNA stem-loop structure (47). Co-expression of the MS2

fusion protein targets the protein of interest to the MS2 stem-loop structure in the target RNA.

This system was used by Kong et al. to demonstrate that human a-globin mRNA is stabilized by

tethering an isoform of murine PCBP2 (murPCBP2-KL) (112). Because the MS2 coat protein

binds to the MS2 stem-loop as a dimer, Hook et al examined the use of a covalent dimer of the

MS2 protein, first described by Peabody and Lim (96, 163). This head-to-tail covalent dimer of

MS2 coat proteins, here termed (MS2)2, results from the in-frame fusion of tandem MS2 open

reading frames by a linker sequence. Using this approach, Hook et al. observed an increase in

specificity and efficacy of the covalent dimer system, relative to that of a single MS2 fusion (96).

For these reasons, we developed a protein-RNA tethering system using the (MS2)2 covalent

dimer to directly examine the role of PCBP2 tethered to the 5'CL in PV RNA replication.

In the case of wild-type PV RNA, PCBP1/2 binds to stem-loop b in the 5'CL and viral

protein 3CD binds to stem-loop d (Figure 3-3A). By replacing the majority of stem-loop b with

an equally sized MS2 stem-loop structure [5'CLMS2] (Figure 3-1C), we removed the PCBP

binding site, thereby preventing endogenous PCBP from binding to the 5'CLMS2. In addition,

expression of the (MS2)2PCBP2 covalent dimer fusion protein, in the presence of the 5'CLMS2

RNA, should tether PCBP2 to the 5'CLMS2 via the interaction of the (MS2)2 covalent dimer with









the MS2 RNA stem-loop structure (Figure 3-3B). By extension, we predict that PV RNA

transcripts containing the 5'CLMS2 will be defective for negative-strand synthesis, and that

co-expression of the (MS2)2PCBP2 fusion protein in the same reaction should restore

negative-strand synthesis definitively showing that PCBP2 is required for negative-strand

initiation.

(MS2)2PCBP2 Binds Specifically to 5'CLMS2 RNA

To establish the functionality of the MS2 protein-RNA tethering system, we first

performed electrophoretic mobility shift assays (EMSA) to examine the protein binding profile

of the 5'CLMS2 relative to that of the 5'CLWT. As expected, the 5'CLWT RNA probe formed a

previously characterized PCBP-5'CL RNP complex (complex I) with either endogenous PCBPs

in HeLa S10 extracts or recombinant PCBP2 (rPCBP2) (Figure 3-4A, lanes 1-4) (12, 77, 158).

In contrast, complex I was not formed in identical binding assays containing the 5'CLS2 RNA

probe (Figure 3-4A, lanes 5-8). This demonstrated that PCBP2 does not bind to 5'CLMS2 RNA

probe, as predicted.

HeLa S10 translation-replication reactions were programmed with a non-translating RNA

(mock translation) or protein expression RNAs which encoded either PCBP2, (MS2)2 or

(MS2)2PCBP2. As expected, the PCBP-5'CL complex (complex I) was formed in binding

assays containing the 5'CLwT RNA probe (Figure 3-4B, lanes 2-5) but not in assays containing

the 5'CLMS2 RNA probe (Figure 3-4B, lanes 7-10). The expression of exogenous PCBP2,

(MS2)2 or (MS2)2PCBP2 had no significant effect on the formation of complex I with the

5'CLWT probe (Figure 3-4B, lanes 3-5). However, the expression of either (MS2)2 or

(MS2)2PCBP2 resulted in the formation of a new, slower-migrating RNP complex (complex II)

with the 5'CLMS2 RNA probe (Figure 3-4B, lanes 9-10). This demonstrated that the

(MS2)2PCBP2 fusion protein binds specifically to 5'CLmS2 RNA and not wild-type RNA.









(MS2)2PCBP2 Restores Negative-strand Synthesis on P23-5'CLMS2 RNA

To ascertain the effectiveness of the MS2 protein-RNA tethering system, we measured the

levels of negative-strand synthesis observed with P23-5'CLMS2 RNA in presence or absence of

(MS2)2PCBP2. Negative-strand synthesis was measured in PIRCs isolated from HeLa S10

reactions that contained either P23 RNA or P23-5'CLMS2 RNA and an equimolar amount of a

protein expression RNA which expressed either (MS2)2, PCBP2 or (MS2)2PCBP2. In the

reactions that contained P23 RNA, the co-expression of (MS2)2 or (MS2)2PCBP2 had little effect

on negative-strand synthesis (Figure 3-5, lanes 1 & 3). There was a detectable increase in

negative-strand synthesis in the presence of exogenous PCBP2 (Figure 3-5, lane 2), which again

is indicative of PCBP2 involvement in negative-strand synthesis. In the reactions containing

P23-5'CLMS2 RNA, negative-strand synthesis was reduced to barely detectable levels in the

reactions that contained either the (MS2)2 or PCBP2 expression RNAs (Figure 3-5, lanes 4-5).

In contrast, a large increase in negative-strand synthesis was observed in the reaction containing

the (MS2)2PCBP2 expression RNA (Figure 3-5, lane 6). In this reaction, negative-strand

synthesis increased approximately 100-fold over the levels observed in the (MS2)2 or PCBP2

control reactions (Figure 3-5, compare lane 6 with 4 & 5). Therefore, these results clearly

established the effectiveness of the MS2 protein-RNA tethering system and showed that PCBP2,

either directly bound or tethered to the 5'CL, is required for efficient initiation of PV

negative-strand RNA synthesis.

Deletion Analysis of PCBP2 Using the (MS2)2 Protein-RNA Tethering System

To define the region of PCBP2 involved in the protein-protein or protein-RNA interactions

relevant to the initiation of negative-strand synthesis, we performed a deletion analysis of

PCBP2, followed by a test of function using the MS2 protein-RNA tethering system. The

PCBP2 coding sequence was divided into three regions, each region containing one of the three









conserved KH domains (Figure 3-6A). The coding sequence for each of these regions was

expressed as an (MS2)2 fusion protein in reactions containing P23-5'CLMS2 RNA. As before,

significant levels of negative-strand RNA synthesis was observed in the presence of

(MS2)2PCBP2 compared to those observed in the (MS2)2 and PCBP2 reactions (Figure 3-6B,

lanes 1-3). Neither expression of the (MS2)2KH1 [Region] nor the (MS2)2KH2[Region] fusion

proteins were able to support negative-strand synthesis above background levels (Figure 3-6B,

lanes 4-5). In contrast, the expression of the (MS2)2KH3 [Region] fusion protein restored

negative-strand synthesis to levels slightly higher than those observed with (MS2)2PCBP2

(Figure 3-6B, lanes 1 & 6). This suggested that the dominant domain in PCB2 that is required

for negative-strand initiation resides in the C-terminal 130 amino acids of PCBP2, which

includes the KH3 domain.

A recent structural analysis of the KH domains of PCBP2 revealed an intramolecular

interaction between the KH1 and KH2 domains that was predicted to influence the function of

one or both of the domains (67). In consideration of this potential effect on the functional

activity, the amino terminal region of PCBP2, including both KH1 and KH2 domains, was fused

to (MS2)2 [KH1/2[Region]], and the replication of P23-5'CLMS2 RNA was measured in the

presence of this fusion protein. In this reaction, negative-strand synthesis was restored to about

70-80% of the levels observed with (MS2)2PCBP2 and about 50-60% of the levels observed with

(MS2)2KH3 [Region] (Figure 3-6C, lanes 1, 4 & 5). Therefore, the results of this replication

assay were consistent with the results of the structural studies which predicted a functional

relevancy for the intramolecular interaction between the individual KH1 and KH2 domains of

PCBP2 (67).









To ensure that the differences in replication efficiency observed in Figure 3-6 were not a

secondary result of variations in the synthesis (or stability) of the (MS2)2 fusion proteins or the

viral replication proteins, we measured the amount of protein synthesized in each reaction.

Labeled proteins were synthesized in HeLa S10 translation reactions containing [35S]-methionine

and were examined by SDS-PAGE and autoradiography (Figure 3-7). The results of this

experiment indicated that all of the (MS2)2 fusion proteins were synthesized in similar amounts

as full-length, intact proteins, and similar levels of the labeled viral proteins were synthesized in

each reaction (Fig. 7). Taken together, these results indicated that the observed differences in

negative-strand synthesis that were a direct result of the efficacy of the given (MS2)2 fusion

protein and not a result of differences in the levels of protein synthesis or protein stability.

The Conserved KH3 Domain is Sufficient to Support Negative-strand Synthesis

Since the C-terminal KH3 containing fragment supported the highest levels of

negative-strand synthesis, we chose to analyze this region further. To determine if the KH3

domain itself was responsible for the observed activity, the residual N- and C-terminal amino

acid sequences outside of the KH3 domain were deleted to form the KH3 [Domain] fusion

protein construct (Figure 3-8A). Separate N- and C-terminal deletions were also made within the

KH3 domain, removing the N-terminal P-strand and the C-terminal a-helix respectively (Figure

3-8A). Due to the structurally conserved nature of the KH domain, these deletions would be

expected to perturb the tertiary structure of the KH3 domain and to disrupt structurally dependent

protein interaction surfaces. The coding sequences for each of these mutants was fused to

(MS2)2, and the replication of P23-5'CLMS2 RNA was measured in the presence of the individual

fusion proteins. Similar levels of protein synthesis were verified as before by SDS-PAGE (data

not shown). As expected, expression of the (MS2)2PCBP2 and (MS2)2KH3 [Region] fusion

proteins supported significant levels of negative-strand synthesis. Removal of the amino acids









flanking the KH3 domain had no inhibitory effect on levels of negative-strand synthesis

observed (Figure 3-8B, lanes 3 & 4), however, deletions in the KH3 domain itself (i.e. KH3AP1

and KH3Aa3) completely inhibited negative-strand synthesis (Figure 3-8B, lanes 5 & 6).

Therefore, the intact KH3 domain was required to support high levels of negative-strand

synthesis.

To confirm that the observed differences in negative-strand synthesis were not due to an

unexpected change in a given (MS2)2 fusion protein's ability to bind to 5'CLMS2 RNA, an EMSA

was performed using 5'CLmS2 RNA probe and each of the PCBP2 fragment (MS2)2 fusion

proteins (Figure 3-9A). In each case, the labeled probe was shifted to form a slower migrating

RNP complex similar to complex II in Figure 3-4B. We also examined the expression and

integrity of each individual (MS2)2 fusion proteins by SDS-PAGE and autoradiography (Figure

3-9B). The results of these experiments clearly showed that the (MS2)2 protein acts as a

functional cassette to efficiently tether a fusion protein to the 5'CL, regardless of that proteins

identity. Additionally, all (MS2)2 fusion proteins appear to be synthesized in the similar amounts

as stable, full-length proteins.

Therefore, these results demonstrate that, when tethered to the RNA, the KH3 domain

alone was sufficient to support initiation of PV negative-strand synthesis. This activity was not

an artifact of increased protein concentration or binding affinity, and from deletion experiments,

this activity appears to be dependent on the intact tertiary structure of the KH domain.

The Combined KH1-KH2 Domain Fragment Does Not Utilize PCBP Dimerization to
Promote Negative-strand Synthesis

PCBP2 dimerization has been shown to be required for PCBP2's function in PV IRES

translation, and an approximate dimerization domain within the KH2 domain was identified (29).

This intermolecular dimerization could potentially be influenced by the previously discussed









intramolecular interaction between the KH1 and KH2 domains (67). In this case, it is possible

that the (MS2)2KH1/2 fusion protein, when tethered to the RNA, forms heterodimers with

endogenous full-length PCBP2 in the HeLa extracts. If so, the negative-strand synthesis

observed in RNA replication reactions could be a result of the function of this additional

molecule of PCBP, rather than a direct function of the KH1/2 domains.

To determine if multimerization with endogenous PCBP was responsible for the observed

activity of the KH1/2 fragment, we deleted the 23 amino acid multimerization domain (MD;

amino acids 125-158 of PCBP2) from the KH2 domain, in the context of the (MS2)2KH1/2

fragment [(MS2)2KH1/2-AMD] (Figure 3-10A) (29). Again, due to the structurally conserved

nature of the KH domain, this deletion would be expected to significantly perturb the tertiary

structure of the KH2 domain. Replication of P23-5'CLMS2 RNA was measured in the presence

of the (MS2)2PCBP2, (MS2)2, (MS2)2KH1/2[Region] and (MS2)2KH1/2-AMD fusion proteins.

Similar levels of protein synthesis were verified as before by SDS-PAGE (data not shown). As

expected, expression of the (MS2)2PCBP2 and (MS2)2KH1/2[Region] fusion proteins supported

relative levels of negative-strand synthesis comparable to previous experiments (Figure 3-10B,

lanes 1 & 3). Surprisingly, deletion of the MD did not abolish the ability of the KH1/2 fusion

protein to promote negative-strand synthesis, although the level of RNA product observed were

decreased slightly. Therefore, the intact KH1/2 region, when tethered to the RNA, does not

require multimerization to promote negative-strand synthesis.

Multiple PCBP Isoforms Support Initiation of Negative-strand RNA Synthesis

Given the high degree of sequence conservation among members of the PCBP family,

particularly within the KH domains, it was likely that multiple PCBP isoforms may share the

ability to promote PV negative-strand initiation. Additionally, it was already known that PCBP1

could bind to the 5'CL and could also restore PV replication in Poly(C)-depleted cell extracts









(77, 209). Further, the dominant splice variant ofPCBP2, PCBP2-KL, only differs by the

exclusion of 31 amino acids encoded by exon 8a, suggesting that PCBP2-KL would likely

function similarly (Figure 3-11A) (76, 128). Together, PCBP1, PCBP2, PCBP2-KL represent

the most closely related and highly abundant members of this protein family (48, 127), and we

would therefore predict that each of these isoforms would function at some level to restore

negative-strand RNA synthesis in the (MS2)2 tethering system.

To determine the efficiency with which PCBP1 could function in this system, we measured

the levels of negative-strand synthesis of P23-5'CLMS2 RNA in the presence of either PCBP1,

(MS2)2PCBP1, PCBP2 or (MS2)2PCBP2. As before, negative-strand synthesis was measured in

PIRCs using equimolar amounts of P23-5'CLMS2 RNA and the individual protein expression

RNA. In the reactions that contained P23-5'CLMS2 RNA, co-expression of PCBP1 or PCBP2

alone was unable to promote negative-strand synthesis (Figure 3-11B, lanes 1 & 3). As

predicted, (MS2)2PCBP1 was able support negative-RNA synthesis, and interestingly, the levels

of negative-strand synthesis were slightly higher (-1.5-fold) than those observed in reactions

containing (MS2)2PCBP2 (Figure 3-11B, lanes 2 & 4).

To determine the efficiency with which PCBP2-KL could function in the (MS2)2 tethering

system, we measured the levels of negative-strand synthesis of P23-5'CLMS2 RNA in the

presence of either PCBP2, (MS2)2PCBP2, PCBP2-KL or (MS2)2PCBP2-KL. Here again,

negative-strand synthesis was measured in PIRCs using equimolar amounts of P23-5'CLMS2

RNA and the individual protein expression RNA. In reactions that contained P23-5'CLMS2

RNA, co-expression of PCBP2 or PCBP2-KL alone was unable to promote negative-strand

synthesis (Figure 3-11C, lanes 1 & 3). As expected, (MS2)2PCBP2-KL was able support levels









of negative-RNA synthesis similar to those observed in reactions containing (MS2)2PCBP2

(Figure 3-11C, lanes 2 & 4).

Not All PCBP Family Members Support Negative-strand synthesis

Although their potential role in PV replication has never been investigated, the more

distantly related members of the PCBP family (PCBP3, PCBP4, PCBP4A, and hnRNP K) are all

expressed to varying levels in different tissue types (128). More importantly, we were able to

detect mRNA corresponding to each protein in HeLa cell total RNA by RT-PCR, indicating the

presence of each of these less abundant isoforms in HeLa cell extracts. HnRNP K is the most

distantly related to all other PCBPs, and undergoes significant nucleo-cytoplasmic shuttling

(135), but is predominantly localized to the nucleus except during cell-cycle signaling (118).

PCBP3 and PCBP4/4A exhibit cytoplasmic localization and appear to be excluded from the

nuclear compartment (48), indicating their availability to participate in cytoplasmic PV

replication. PCBP4A is a splice variant of PCBP4, differing only in the carboxy-terminal amino

acids, but contains an identical KH1, KH2, variable region, and 90% of KH3 (128). Despite a

higher degree of amino acid similarity within individual KH domains, PCBP3/4/4A are

significantly divergent from PCBP1/2 and may not retain all necessary functions relative to PV

replication (Figure 3-12A).

To determine the relative abilities of the various PCBP family members to function in the

(MS2)2 tethering system, we measured the levels of negative-strand synthesis of P23-5'CLMS2

RNA in the presence of either (MS2)2, (MS2)2PCBP1, (MS2)2PCBP2, (MS2)2PCBP2-KL,

(MS2)2PCBP3, (MS2)2PCBP4, (MS2)2PCBP4A, or (MS2)2hnRNP-K. As before,

negative-strand synthesis was measured in PIRCs using equimolar amounts of P23-5'CLMS2

RNA and the individual protein expression RNA. As before, (MS2)2PCBP1, (MS2)2PCBP2, or

(MS2)2PCBP2-KL were all able to promote negative-strand RNA synthesis of P23-5'CLMS2









RNA to similar levels (Figure 3-12, lanes 2-4). The (MS2)2 fusion of the most distantly related,

predominantly nuclear hnRNP K was unable to support significant levels of negative-strand

synthesis, as may have been expected (Figure 3-12, lane 8). Surprisingly, the distantly related

PCBP4, and to a lesser extent PCBP4A, functioned better than PCBP1, 2, or 2KL, whereas the

more closely related PCBP3 appeared to support very modest levels of negative-strand RNA

synthesis (Figure 3-12, lanes 5-7).

To further determine if these more distantly related PCBPs could plausibly be involved in

natural PV replication, the ability of each of the isoforms was assayed for its ability to bind the

wild-type 5'CL. RNA binding was ascertained by EMSA using a 5'CL riboprobe and bacterially

recombinant PCPBs as described in Chapter 2. Since the splice variants PCBP2-KL and

PCBP4A maintain the same RNA binding determinants as their parental proteins, only PCBP2

and PCBP4 were assayed. As previously observed, both PCBP1 and PCBP2 were able to form

RNP complexes with the PV 5'CL, and corresponding with its significant divergence, no RNP

complex was formed in the presence of hnRNP K (Figure 3-13, lanes 2, 3, & 6). Interestingly,

both PCBP3 and PCBP4 were able to form RNP complexes with the PV 5'CL (Figure 3-13,

lanes 4 & 5), suggesting that these isoforms have the potential to, if present, form an RNP

complex with viral RNA and participate in negative-strand initiation.

Discussion

The work presented here demonstrated the requirement for PCBP in the initiation of PV

negative-strand synthesis. Furthermore, we established that a direct PCBP-RNA interaction was

not required to mediate this function by developing and using the (MS2)2 protein-RNA tethering

system to investigate PV negative-strand synthesis. We demonstrated the utility of this system in

analyzing regions in an essential cellular protein, relative to PV replication, without affecting

other viral and cellular processes in which the protein is involved. In doing so, we have shown









the KH3 domain of PCBP2, when tethered to the RNA, was able to support the initiation of

negative-stand RNA synthesis. This suggests that a structurally conserved protein-protein or

protein-RNA interaction surface which is required for negative-strand initiation exists within this

conserved domain. We have also noted the functional redundancy of the combined KH1/2

domains of PCBP2 relative to PV RNA replication, consistent with recent work demonstrating

the in vitro functionality of PCBP2 with the KH3 domain deleted (165).

Prior Indications of PCBP Involvement in Poliovirus RNA Replication

Initial in vivo studies identified an RNP complex formed at the 5' end of PV genomic

RNA which appeared to be involved at some stage of RNA replication (13). Further

investigation of this complex revealed the presence of both a viral (3CD) and cellular (PCBP2)

protein, and showed that disruption of this complex inhibited RNA replication (12, 158). Further

in vitro analysis using a PCBP binding site point mutant (C24A) revealed an additional role for

PCBP in PV RNA stability (144). Using an alternative approach, Walter et al. showed that

replication of a dicistronic PV RNA replicon was inhibited in cell extracts which were depleted

for PCBPs (209). However, the above work was either unable to directly account for effects on

RNA stability or to differentiate between defects in negative- and positive-strand synthesis.

Our results showed that the C24A mutation, which inhibits PCBP binding, also inhibits

negative-strand synthesis without inhibiting positive-strand synthesis. Using a trans-replication

assay, we showed definitively that this defect was not a secondary effect of a deficiency in either

protein synthesis or RNA stability. Therefore, these findings suggested but did not prove that

PCBP is a co-factor in the initiation of PV negative-strand synthesis.









(MS2)2 Protein-RNA Tethering Assay Demonstrated that PCBP is Required for Poliovirus
Negative-strand Synthesis

We directly addressed the role of PCBP2 in the initiation of PV negative-strand synthesis,

by developing a protein-RNA tethering system. This system utilized the high affinity interaction

ofMS2 bacteriophage coat protein with its cognate RNA stem-loop (47, 58). We modified this

system as described by Hook et al. to take advantage of the added specificity and efficacy

conferred by using an MS2 protein covalent dimer, (MS2)2 (96). By replacing the natural PCBP

binding site in the 5'CL of PV genomic RNA with the MS2 stem-loop (5'CLMS2), we were able

to target the (MS2)2PCBP2 fusion protein to the 5'CLMS2 RNA.

This system has many significant advantages over other approaches used to functionally

characterize cellular protein involvement in virus replication. It is very difficult to isolate

individual functions of multi-functional cellular proteins using techniques such as RNAi, gene

knockout, protein depletion and dominant-negative inhibition, which can result in a broad

spectrum of downstream effects unrelated to the function of interest. In addition, some of these

techniques are not feasible in certain systems, while others present significant technical

challenges. The (MS2)2 protein-RNA tethering system could be adapted for in vivo and in vitro

use, and functional analysis with this system can be performed with minimal disruption of other

normal cellular processes. In the PV life-cycle, PCBP2 is used in both IRES-dependent

translation and RNA replication. Our analysis of PCBP2's role in replication using the (MS2)2

protein-RNA tethering system can be performed without affecting PCBP2 binding to the IRES,

since the fusion protein is targeted specifically to the 5'CL. The system also permits us to

precisely define the protein bound to the 5'CL, thereby providing a platform to perform

mutagenic analysis of protein function in a straightforward manner.









Using the (MS2)2 protein-RNA tethering system, we observed a significant defect in

negative-strand synthesis of a 5'CLMS2 template RNA, concurrent with the loss of PCBP binding

to the 5'CLMS2. We then demonstrated a restoration of negative-strand synthesis upon

co-expression of the (MS2)2PCBP2 protein. This conclusively showed that the presence of

PCBP2 at the 5'CL of PV genomic RNA is required for the initiation of negative-strand

synthesis. Furthermore, PCBP's function is not mediated through direct protein-RNA interaction

with the 5'CL, since tethered PCBP2 was capable of restoring RNA replication.

The requirement for PCBP in negative-strand initiation is consistent with the current model

of PV replication complex formation involving genome circularization mediated by

protein-protein interactions between RNP complexes formed at the 5' and 3' ends of PV

genomic RNA (26, 93, 126, 196). By this model, PCBP2 bound or tethered to the 5'CL interacts

with PABP bound to the poly(A) tail, thereby circularizing the genome and allowing the

subsequent initiation of negative-strand synthesis.

The Combined KH1 & KH2 Fragment or the KH3 Domain of PCBP2 is Required for
Negative-strand Initiation

An important application of the tethered function assay is mutational analysis of the

tethered protein to determine the regions involved in protein-protein interactions. This illustrates

yet another advantage of this system in that functional analysis of PCBP fragments can be

performed without requiring direct binding to the 5'CL. Using this system, we were able to

show that the KH3 domain of PCBP2 contains sequences and structures sufficient for the

functional interactions involved in the initiation of negative-strand RNA synthesis. Based on the

current circularization model, the key protein-protein interactions would be between PCBP,

PABP and 3CD, however it is also possible that other proteins are involved, or that PCBP

interacts with an as yet unknown RNA element in the 3' end of the viral genome.









We were also able to observe a functional redundancy in PCBP2 residing in the

amino-terminal KH1 and KH2 domains that was capable of mediating similar critical

interactions. This function was not observed when either the KH1 or KH2 domains were used

separately, consistent with recent NMR structural data, which indicated structural differences

between the PCBP2 KH1 or KH2 domains individually and a tandem KH1/2 construct (67).

Multiple studies from Du et al. have identified large hydrophobic faces on KH1 and KH2 which

would allow the two domains to interact intramolecularly, further intertwining the function of the

KH1 and KH2 domains (67, 68). These results are also consistent with the recent work by

Perera et al. which demonstrated the ability of PCBP2 with a KH3 deletion to restore PV RNA

replication in Poly(C)-depleted cell extracts (165). A dimerization domain in PCBP2 has been

identified and it was shown that this sub-domain within KH2 was required for IRES function

(29). This domain was deleted from the KH1/2 fusion protein and was shown to be dispensable

for negative-strand synthesis, indicating that the activity of KH1/2 fragment does not require

multimerization.

The fully functional KH3 fragment does not contain any established dimerization

sequences; so here again, dimerization of PCBP2 does not appear to be required to mediate the

interactions required for negative-strand initiation. This does not rule out the possibility that

dimerization of PCBP may be involved in physiologic PCBP binding to the 5'CL, since this

interaction has been bypassed using the protein-RNA tethering system.

A Subset of PCBP Isoforms Support Initiation of Negative-strand RNA Synthesis

The PCBPs represent a family of poly(rC/dC) binding proteins which include hnRNP K

and PCBPs 1-4, and two additional predominant splice variants, PCBP2-KL and PCBP4A (109,

121, 128, 133). This protein family is characterized by the presence and positioning of three

highly homologous KH domains (80, 188), which were originally characterized as RNA binding









domains (65, 67) but have been evolutionarily retuned to perform additional or alternative

functions. Given the high degree of sequence conservation among members within the PCBP

family, particularly between corresponding KH domains, it was likely that some functional

overlap between different isoforms of PCBP. Specifically, different PCBP isoforms could share

the ability to promote PV negative-strand initiation. Both PCBP1 and PCBP2 are able to bind to

the 5'CL and were able restore PV replication in poly(C)-depleted cell extracts (77, 209). Since

PCBP2 and its splice variant PCBP2-KL share the same RNA binding determinants and only

differ by 31 amino acids, it is likely that PCBP2-KL functions similarly (76, 128). Through the

application of the (MS2)2 protein-RNA tethering system, we have shown that PCBP1, PCBP2,

and PCBP2-KL all share similar functionality relative to PV RNA replication and can support

similar levels of negative-strand synthesis.

Despite a higher degree of amino acid similarity within individual KH domains, PCBP3,

PCBP4, and PCBP4A are significantly divergent from PCBP1/2/2KL and may not retain all

necessary functions relative to PV replication. Even within the KH domains, hnRNP K is clearly

the most divergent and distantly related PCBP isoform, and would be predicted to share very

little functional similarity with the other family members (128). We have shown by RT-PCR

that the more distantly related and less abundant PCBP isoforms are in fact expressed in HeLa

cells, and have further shown that PCBP3 and PCBP4/4A, but not hnRNP K, are capable of

forming critical RNP complexes with the PV 5'CL. Again, using the (MS2)2 tethering system,

we have shown that PCBP4 and PCBP4A are capable of supporting higher levels of

negative-strand synthesis than the levels supported by PCBP1/2/2KL. Interestingly, PCBP3 was

only able to support minimal levels of negative-strand synthesis, suggesting that PV RNA

replication may be inhibited in cells which express high levels of PCBP3. Taken together this









suggests that during natural PV infection, PCBP3, PCPB4 or PCBP4A would each possess the

ability to bind to the 5'CL and incorporate into the RNP complexes critical for initiation of

negative-strand RNA synthesis. Furthermore, PCBP3 could potentially act as an inhibitor of

viral RNA replication by forming a non-functional RNP complex with the 5'CL of PV RNA.











A.





5' Cloverleaf
(5'CL)





B.





5' Cloverleaf C24A
(5'CLC24A)


| Stem-loop 'c'
AC G
NP( I3'-hirld4in_ site C U 3CDpro-binding site
S C-G
CC CACCCCAGAG GGGUA CCGGUAU UG
1A I I 1 11 1I I 1 I 1 11
U G UUGGGGUCUC CCG CAUGUCCCAUG GC
| Stem-loop'b' C-G I Stem-loop'd
A-U
A-iL
A-U Stm 'a
A-U
U-A
5'-U UAS3
5- -U A-3'


A G
. ..' .....i C U 3CDpro-binding site
C-G

C ACCCCAGAG GGCAGUA CGGUAU U G
A I III I1 l] I II
U UlGGGGLICLIC CCG CAUGUCCCAUG GC
GU GA-U U
C-G
A-U
A-U
A- U

5'-U U-UA-3'


C. ACG
(MS2)-binding site C U 3CDpr'-binding site
I I C-G
G-CG UA ICC UU
UA CC CAUGUAGG- CGGCUA GUACCGGUA G
1 1I I I I II I I l l I I I I I I I I
UAGGAGUACArLC CCG C"LIGu UCCCAUG GC
GA-U U
5' Cloverleaf MS2 C-G
A-U
(5'CLMs2) A-U
A-U

5- UUA -3'
Figure 3-1. Diagrams of the wild-type and mutant 5' cloverleaf. A) The PV 5'CL is divided into
four domains: stem a, and stem-loops b, c, and d. A region of stem-loop b functions
as the PCBP binding site, whereas 3CDpro binds to structural elements in stem-loop d.
B) The 5'CLC24A contains a single mutation (indicated in red) in the PCBP binding
site of stem-loop b. C) The 5'CLMS2 has had the majority of stem-loop b replaced
with the MS2 bacteriophage coat protein binding site (MS2 stem-loop).









A.


5' CL"T


5' CL14


B.


6)


1 2 3 4 5 6

Figure 3-2. The C24A mutation inhibits PCBP binding and negative-strand RNA synthesis.
A) Electrophoretic mobility shift assay (EMSA) using radiolabeled RNA probes,
either 5'CLWT RNA (lanes 1-3) or 5'CLC24A RNA (lanes 4-6). The RNA probe was
either run alone (lanes 1 & 4), with HeLa S10 extracts (lanes 2 & 5), or with
bacterially expressed rPCBP2 (lanes 3 & 6). The specific RNP complex formed with
the 5'CLWT RNA probe and cellular PCBP is labeled as complex I. B) Replication of
P23 RNA and P23-5'CLC24A RNA was measured using PIRCs isolated from HeLa
S10 reactions. Radiolabeled product RNA was visualized by denaturing
CH3HgOH-agarose gel electrophoresis and autoradiography. These P23 RNA
transcripts allow only negative-strand synthesis due to the presence of two non-viral
G residues at the 5' end as a result of T7 transcription.










5' Cloverleaf
(5'CL)


B.


5' Cloverleaf MS2
(5'CLMS2)


(MS2)PCBP2
I+(MS),PCBP2


5 3'
::iP
51 "


Figure 3-3. Schematic of the (MS2)2 protein-RNA tethering system. A) Schematic of the
wild-type 5'CL RNP complex. This complex consists of PCBP bound to stem-loop b
and 3CDpr' bound to stem-loop d. B) Schematic of the 5'CLMS2 RNP complex.
Endogenous PCBP in cell extracts (or recombinant PCBP) is no longer able to bind to
the 5'CL because stem-loop b has been replaced with the MS2 stem-loop. In the
absence of PCBP binding, 3CDpro can still bind to the 5'CLMS2, but at lower affinity.
When the (MS2)2PCBP2 fusion protein is provided, it is recruited to the 5'CLMS2 via
the (MS2)2 interaction with its cognate stem-loop. This effectively tethers PCBP2 to
the 5'CL, forming a surrogate 5'CL RNP holocomplex.


A.












A.


5' CL"' 5' CL"S
-* lips


B. # A


5' CL"T 5' CL"Is2
ruse Con$


B.4.

*gh'


1 2 3 4


5 6 7 8


1 2 3 4 5


6 7 8 910
6 7 8 9 10


Figure 3-4. The 5'CLMS2 binds (MS2)2 fusion proteins but does not bind PCBP2.
A) Electrophoretic mobility shift assay (EMSA) using radiolabeled RNA probes,
either 5'CLwT RNA (lanes 1-4) or 5'CLMS2 RNA (lanes 5-8). The RNA probe was
either run alone (lanes 1 & 5), with HeLa S10 mock translation reactions (lanes
2 & 6), with bacterially expressed rPCBP2 (lanes 3 & 7) or a vector control bacterial
extract (lanes 4 & 8). The specific RNP complex formed with the 5'CLWT RNA
probe and endogenous cellular PCBP is labeled as complex I. B) EMSA using either
5'CLwT RNA probe (lanes 1-5) or 5'CLMS2 RNA probe (lanes 6-10). The probe was
either run alone (lanes 1 & 6), with a HeLa S10 mock translation reaction (lanes
2 & 7), or with HeLa S10 translation reactions in which the indicated proteins were
expressed (lanes 3-5, 8-10). Specific RNP complexes were formed with the 5'CLWT
RNA and endogenous cellular PCBP (complex I), or with the 5'CLMS2 RNA and
(MS2)2 or (MS2)2PCBP2 (complex II).
























P23 P23-5'CLm2




1 2 3 4 5 6

Figure 3-5. The (MS2)2PCBP2 fusion protein restores negative-strand synthesis of a 5'CLMS2
RNA template. Negative-strand synthesis was measured in reactions containing
either P23 RNA (lanes 1-3) or P23-5'CLMS2 RNA (lanes 4-6) using PIRCs isolated
from HeLa S10 reactions. Each reaction contained either P23 RNA or P23-5'CLMS2
RNA and an equimolar amount of a protein expression RNA which expressed either
(MS2)2, PCBP2 or (MS2)2PCBP2, as indicated. Both template RNAs were capped to
ensure equal template stability. Radiolabeled product RNA was visualized by
denaturing CH3HgOH-agarose gel electrophoresis and autoradiography









PCBP2 N-B U-- A a1-C
KHl1 KH2 KH3


N-1- --C
KH1

N-K C
KH2

N-N -C
K31

N-E U-E I C
Kill KH2


4N
S


1 2 3 4 5 6


N


2 3 4 5


Figure 5-6. Identification of the functional domains within PCBP2 that restore negative-strand
RNA synthesis of a 5'CLMS2 template RNA. A) Schematic of the domain structure of
PCBP2, including the conserved KH1, KH2 and KH3 domains. Each PCBP2 region
depicted was fused to (MS2)2 and assayed in replication reactions.
B & C) Negative-strand synthesis was measured in reactions containing P23-5'CLMS2
RNA and an equimolar amount of an RNA which expressed the indicated RNA. The
total molar RNA concentration and molar RNA ratio were maintained in each
reaction, and the input template RNA contained a 5' cap.


A.


KH1
Region

KH2
Region

KH3
Region

KH1/2
Region


B.


C.


/
AQN


S
I


N
aj

c3r c7





















3CD- 411--1._

2BC- ""


a- --..


2A-


1 2 3


4 5 6 7


Figure 3-7. Levels of protein synthesis observed in the (MS2)2 protein-RNA tethering
replication reactions. HeLa S10 translation reactions which correlate to those
described in Figure 5-6 were incubated with [35S]methionine to metabolically label all
newly synthesized protein products. The labeled proteins synthesized in these
reactions were analyzed by SDS-PAGE and autoradiography. Each reaction
contained an equimolar amount of P23-5'CLMS2 RNA and the indicated (MS2)2
fusion protein expression RNA.


Vlp- -sAi









N-I *-- --- -C
PCBP2 N- -C
KH1 KH2 KH3


KH3 Apl


KH3 Aa3











f1
1


NE NC


NE MC
KH3
A


KH3


2 3 4


5 6


Figure 3-8. Characterization of the KH3 domain using the (MS2)2 protein-RNA tethering
system. A) Schematic of PCBP2 and the KH3 domain deletion mutants used in this
experiment. B) Negative-strand synthesis was measured using PIRCs isolated from
HeLa S10 reactions containing P23-5'CLMS2 RNA and an equimolar amount of a
protein expression RNA as indicated above. The total molar RNA concentration and
molar RNA ratio were maintained in each reaction, and the input template RNA
contained a 5' cap.


A.


KH3
Region

KH3
Domain


N -K U-C
KH3


B.









A.


5.IN
NO .0

o ,P CI ^4^>t"at^~j~"


Sm


LLt-


P 1 2 3 4 5 6 7


lw
*No


8 9 10


aM ..-*o


1 2 3 4 5 6 7 8 9 10


Figure 3-9. The (MS2)2 fusion proteins are evenly expressed, stable, and bind to 5'CLMS2 with
similar affinity. A) An EMSA was performed using a radiolabeled 5'CLMS2 RNA
probe. The probe was either run alone (lane 1), with a HeLa S10 mock translation
reaction (lane 2), or with HeLa S10 translation reactions in which the indicated
proteins were expressed (lanes 3-11). B) Portions of the same HeLa S10 translation
reactions used above were incubated with [35S]methionine, and the labeled protein
products were analyzed by SDS-PAGE and autoradiography.


B.


4


%W









A.


PCBP2


KH1/2
Region


N- W -- U K --C
KH1 KH2 KH3


N-E U-- M- C
KH1 KH2


A
KHI/2 N-I I--[ m C
AMD KHl KH2







B.







1 2 3 4

Figure 3-10. The ability of the combined KH1/2 domains to restore negative-strand synthesis
does not require the multimerization domain. A) Schematic of PCBP2, KH1/2 region,
and the multimerization domain deletion mutant used in this experiment. B)
Negative-strand synthesis was measured using PIRCs isolated from HeLa S10
reactions containing P23-5'CLMs2 RNA and an equimolar amount of a protein
expression RNA as indicated above. The total molar RNA concentration and molar
RNA ratio were maintained in each reaction, and the input template RNA contained a
5' cap.









A.


PCBP1

PCBP2


PCBP2-KL


No.
Amino Acids

C 356

C 365

C 331


B.


1 2 3


4 5


C.


3. j H I B
1 2 3 4 5

Figure 3-11. PCBP1, PCBP2, and PCBP2-KL restore negative-strand synthesis to similar levels
in the (MS2)2 protein-RNA tethering system. A) Schematic of the domain structure
of PCBP1, PCBP2 and PCBP2-KL. Each PCBP isoform depicted, as well as its
corresponding (MS2)2 fusion protein, was assayed in replication reactions.
B & C) Negative-strand synthesis was measured in reactions containing P23-5'CLMS2
RNA and an equimolar amount of an RNA which expressed the indicated protein.
The total molar RNA concentration and molar RNA ratio were maintained in each
reaction, and the input template RNA contained a 5' cap.


/


zp









A.

PCBP1

PCBP2

PCBP2-KL

PCBP3

PCBP4

PCBP4A

hnRNP-K





B.


No.
Amino Acids
356

365

331

339


> 403
C
329

463


1 2 3 4 5 6 7 8

Figure 3-12. PCBP4/4A, but not PCBP3 or hnRNP K, restores negative-strand synthesis in the
(MS2)2 protein-RNA tethering system. A) Schematic of the domain structure of the
PCBPfamily. Each PCBP isoform depicted was fused to (MS2)2 and assayed in
replication reactions. B) Negative-strand synthesis was measured in reactions
containing P23-5'CLMS2 RNA and an equimolar amount of an RNA which expressed
the indicated protein. The total molar RNA concentration and molar RNA ratio were
maintained in each reaction, and the input template RNA contained a 5' cap.













J4
2


Complex I (


1 2 3 4 5 6


Figure 3-13. All PCBP family proteins, except hnRNP-K, bind to the PV 5'CL. A) An EMSA
was performed using radiolabeled 5'CLWT RNA probe and clarified bacterial
recombinant protein extract. The RNA probe was incubated with a vector only
expression control (lane 1) or with a bacterially expressed recombinant PCBP isoform
as indicated (lanes 2 & 6). The specific RNP complexes formed with the 5'CLWT
RNA probe and the various PCBP is isoforms are indicated as isotypes of complex I









CHAPTER 4
2BC-P3 IS THE CRITICAL CIS-ACTING VIRAL PROTEIN PRECURSOR DIRECTING
INITIATION OF POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS

Introduction

Each stage of the viral life-cycle must be carefully orchestrated, both spatially and

temporally, to optimize total virus yield. The evolutionary imperative to do so is met by a

myriad of obstacles at every step, yet viruses have developed multiple mechanisms to overcome

these challenges and replicate efficiently. One common theme among these adaptations is a

close coupling between sequential steps of viral replication; predicating the initiation of one step,

not simply on completion of the previous step, but also on the spatial availability and

functionality of the products of that previous step. The primary means by which many viruses

accomplish this is through extensive rearrangement of the host cell architecture and the creation

of structures known as virus inclusions or virus factories (reviewed by (145)). While the creation

of these structures is well established, the mechanisms which drive the coupling of the viral

life-cycle i/ i/hi/ these structures continue to be of great interest.

Poliovirus infection has been shown to causes dramatic membrane rearrangements

resulting in the formation of characteristic vesicles within the host cell cytoplasm (44). For PV,

the cytoplasmic surface of these membrane vesicles serves as the site of viral replication

complex assembly, RNA replication, and infectious particle assembly (34, 35, 43-45, 51, 167).

Recent data from Egger & Bienz suggest a tight coupling between viral translation at the

endoplasmic reticulum and the formation of these membrane vesicles as well as concurrent

replication complex formation (70, 71). Furthermore, PV defective interfering particles all

maintain the correct reading frame and harbor deletions within the capsid coding region,

suggesting that active translation and RNA replication are linked (85, 115). This coupling was

further substantiated by Novak and Kirkegaard who showed that cis translation of mutant viral









RNA is a prerequisite to the replication of that particular RNA (151). Additionally, RNA

replication is functionally coupled to infectious particle assembly, such that only newly

synthesized positive-strand virion RNA is encapsidated efficiently (152). Taken together, these

studies depict a highly organized and well coordinated process by which PV infection

progresses, each step inextricably tied to the initiation of the next, in an effort to maximize

replication efficiency and virus yield.

While it is clear that PV does adhere to a tightly coupled replication strategy, the various

molecular mechanisms by which this coupling occurs remain unclear. In an effort to

characterize some of these mechanisms, we utilized the HeLa S10 translation-replication system

to probe the relationship between viral translation and initiation of RNA replication. To do this,

we performed trans-complementation of PV subgenomic RNAs expressing discrete polyprotein

precursors and assayed for the ability of these subgenomic RNAs to serve as templates for

negative-strand RNA synthesis. Herein, we show that the coupling of translation to subsequent

negative-strand synthesis is not simply due to the act of translation itself, but the function of a

specific gene productss. Furthermore, we identify the critical regions) of the PV polyprotein as

that which includes 2B and 3DP"o proteins and/or their precursors. This data, in combination with

previous observations, strongly indicates that it is the translation of the 2BC-P3 precursor in cis

that drives template selection for membrane associated negative-strand RNA synthesis.

Results

To determine if the marked coupling of translation and RNA replication observed during in

vivo PV infection was recapitulated in cell-free replication reactions, we performed a series of

trans complementation experiments utilizing the HeLa S10 translation-replication system. In all

cases, two RNAs were present in these reactions: the template RNA, which acts as the RNA









replication template, and the helper RNA, which acts as the trans protein provider but does not

itself act as a replication template.

Efficient PV Negative-strand Synthesis Requires Translation of Viral Template RNA

We and others have established that neither the capsid proteins nor the capsid protein

coding region is required for PV genome translation and RNA replication both in vitro and in

vivo (61, 85, 106, 115). With this in mind, the template RNA used was either a subgenomic

RNA which expressed all PV replication proteins [P23 RNA] or a similar RNA containing a

frameshift mutation and subsequent early termination codons [FS23 RNA] (Figure 4-1A). In

both cases, a full-length PV helper RNA was included in the reaction to provide all PV proteins

and polyprotein precursors in trans. This full-length helper RNA contains a five nucleotide

deletion in the 3' NTR which has been previously shown to inhibit negative-strand synthesis

without affecting translation [PV1AGUA3 RNA] (26). In all cases, detected product RNA

represents only negative-strand synthesis as a result of two non-viral G residues at the 5'end of

all template RNAs used in this study.

PV negative-strand RNA synthesis was assayed using preinitiation replication complexes

(PIRCs) isolated from HeLa S10 translation-replication reactions as previously described in

Chapter 2. Equimolar amounts of template RNA [P23 or FS23] and helper RNA [PV1AGUA3]

were co-translated and subsequent negative-strand synthesis was measured by [a-32P]CTP

incorporation and visualized by denaturing CH3HgOH-agarose gel electrophoresis and

autoradiography. As shown in Figure 4-1B, negative-strand synthesis from the non-translating

FS23 template RNA was reduced 5 to 10-fold relative to that observed from the P23 template

RNA, which translated all replication proteins in cis. As expected, there were slightly increased

levels of the viral P23 proteins produced in the reaction containing both P23 and PV1AGUA3









RNAs relative to that produced in the reaction containing the non-translating FS23 RNA.

However, in reactions in which the amount of helper RNA was doubled to approximate reaction

conditions with elevated replication proteins, the levels of negative-strand synthesis in reactions

containing FS23 RNA remained significantly lower than those observed with P23 RNA (data not

shown). Additionally, if proteins coming in cis and in trans contributed equally to RNA

replication, one would expect the levels of negative-strand synthesis supported by proteins

coming only in trans to be half of that observed when proteins are provided both in cis and in

trans, rather than the 10-15% that was experimentally determined (Figure 4-1B, compare lanes

1 & 2). Therefore, these data indicate that the observed difference in negative-strand synthesis

between P23 and FS23 RNA templates was predominantly due to the translation status of the

RNA being replicated.

In many translation systems, RNAs harboring premature termination codons or RNAs that

are translationally inactivated are often subject to nonsense mediated decay or other forms of

RNA degradation (56, 146). To determine if the observed negative-strand synthesis defect of

FS23 was an indirect effect of RNA instability we performed RNA stability assays under

conditions identical to those used to assay for negative-strand RNA synthesis. Radiolabeled

input RNA, either P23 or FS23 RNA, was co-translated with PV1AGUA3 RNA and the amount

of input RNA remaining was assessed at various times by CH3HgOH-agarose gel electrophoresis

and autoradiography. There was no detectible defect in the stability of FS23 input RNA relative

to P23 RNA (Figure 4-1C, compare lanes 1-4 to lanes 5-8), despite premature translation

termination on FS23 RNA. This result confirmed that the difference in the levels of

negative-strand synthesis observed between P23 and FS23 RNA templates was not due to a

secondary stability defect arising from the absence of elongating ribosomes.









Template RNA Translation Alone is Not Sufficient to Promote Efficient PV
Negative-strand RNA Synthesis

To determine if translation of an entire reading frame and/or translation termination in the

authentic context was required for efficient initiation of negative-strand synthesis, a PV1

subgenomic RNA was constructed in which the sequences coding for 2A through 3C and a

portion of 3D were deleted (P1-3D* RNA; Figure 4-2A). This RNA is actively translated to

produce a fusion of the P1 capsid precursor protein and a carboxy terminal fragment of 3D, but

does not produce any active 3DP"o, and terminates translation in the authentic context. A

derivative of this subgenomic RNA was then constructed by the addition of a frameshift

mutation at nucleotide 1119 of P1-3D* RNA (FS1-3D* RNA; Figure 4-2A). Translation of this

RNA initiates properly but terminates prematurely, resulting in synthesis of a truncated protein

product and incomplete ribosomal transit of the template RNA.

Negative-strand RNA synthesis was assayed as described above in reactions containing

either P1-3D* or FS1-3D* template RNA and an equimolar amount of PV1AGUA3 helper RNA.

As shown in Figure 4-2B, no significant difference in the levels of negative-strand synthesis was

observed between reactions containing P1-3D* or FS1-3D* RNA. Additionally, the amount of

negative-strand synthesis observed from both reactions is significantly reduced from those

observed from P23 RNA which translates all viral replication proteins in cis (data not shown).

These data clearly show that neither complete ribosome translocation through a template RNA,

nor termination of translation in the authentic context, is sufficient to direct efficient

negative-strand synthesis from that template. Furthermore, this result strongly suggests that the

previously observed cis-translation enhancement of negative-strand synthesis is primarily due to

the activity of a viral translation product in cis.









Translation through the 3D Coding Region in cis is Necessary for Efficient PV
Negative-strand Synthesis

To determine which viral protein product or products were required in cis for efficient PV

negative-strand synthesis, a series of subgenomic template RNAs was constructed based on the

P23 RNA used above. For each construct, two stop codons were inserted after the terminal

amino acid residue of the desired protein within the context of the entire polyprotein coding

region of P23 RNA (Figure 4-3A). These constructs all maintained the identical RNA sequences

and structures (with the exception of the stop codons) as the parent P23 RNA, but translated only

a defined amino-terminal portion of the PV polyprotein. Translation of these RNAs initiated

with 2A and progressed normally until reaching the inserted stop codons, whereby P23-2ASTOP

translated 2A, P23-2BSTOP translated 2AB, P23-2CSTOP translated 2ABC, and so forth (Figure

4-3A). In all reactions, the full-length PV helper RNA provided all PV proteins and naturally

occurring polyprotein precursors in trans.

Negative-strand synthesis was assayed as described above from reactions containing one of

the template RNAs depicted in Figure 4-3A with an equimolar amount of PV1AGUA3 helper

RNA. Interestingly, RNA templates which translated anything less than the complete P23

polyprotein exhibited a significant defect in their ability to support efficient negative-strand

synthesis (Figure 4-3B, compare lanes 1-6 to lane 7). The replication deficient P23-3CSTOP RNA

and the replication competent P23 RNA only differ by the inclusion of the 3D coding region,

implicating 3DP"o or a 3D-precursor as the cis protein requirement. Since the majority of the 3D

coding region was present in the replication deficient P1-3D* RNA (Figure 4-2), it is highly

unlikely that physical ribosome transit through this region is responsible for the observed effect.

Taken together, these data strongly indicate that efficient PV negative-strand synthesis requires

cis translation of 3Dpol, 3CDPro, and/or another 3D containing precursor.









Translation of the 2BCP3 Protein Precursor in cis is Sufficient for Efficient PV
Negative-strand Synthesis

To determine if efficient PV negative-strand synthesis requires cis translation of 3DP"o

alone or the translation of a larger 3D containing precursor, a second series of RNA templates

was constructed. These constructs all contain the same 5' and 3' NTRs, however each RNA in

the series contains the coding sequence for a successively larger 3D containing precursor that has

been previously associated with PV replication (Figure 4-4A) (104, 119, 159). In addition to

these template RNAs, an additional control RNA was generated which contained an in-frame

deletion of nucleotides 867-6011 from full-length PV1 RNA [PVlp50 RNA]. This RNA retains

the entire PV 5' and 3' NTRs, utilizes the authentic start/stop codons and codon contexts, and

translates a non-functional 50 kDa protein (p50), serving as an additional control for the effect of

ribosome transit through the RNA. Here again, the full-length PV helper RNA provided all PV

proteins and naturally occurring polyprotein precursors in trans.

Negative-strand synthesis was assayed as described above from reactions containing one of

the template RNAs depicted in Figure 4-4A with an equimolar amount of PV1AGUA3 helper

RNA. As shown in Figure 4-4B, RNAs which translated p50, 3Dpo1, 3CDpro, 3BCD, or P3

proteins in cis were all unable to efficiently serve as RNA templates for negative-strand synthesis

(Figure 4-4B, compare lanes 1-5 to lane 7). Strikingly, template RNA which translated the

2BC-P3 precursor protein in cis supported negative-strand synthesis to levels greater than that

observed with P23 RNA (Figure 4-4B, compare lane 7 to lane 8). These data clearly

demonstrate that neither cis translation of 3DP"o alone nor ribosomal transit through the 3D

coding region of a template RNA is sufficient to promote efficient PV negative-strand synthesis

on that template. The replication deficient P3 RNA and the replication competent 2BC-P3 RNA

only differ by the inclusion of the 2BC coding region, which indicates strongly that efficient PV









negative-strand synthesis requires cis translation of either the full 2BC-P3 precursor protein or a

combination of individual, discrete protein functions within the 2BC-P3 polyprotein.

If efficient PV negative-strand synthesis requires cis translation of multiple discrete

proteins/precursors, one of the most likely candidate proteins (in addition to 3DPol/3CDprO) is

protein 2C. Protein 2C has been directly implicated in negative-strand initiation and has been

shown to specifically bind to PV RNA (17, 18, 122, 170, 202). Therefore, a 2C-P3 expressing

template RNA was constructed and tested as was described above (Figure 4-5A), despite the fact

that a 2C-P3 precursor protein has neither been observed nor postulated to play a role in PV

replication. Negative-strand synthesis was assayed as described above from reactions containing

P3, 2C-P3, or 2BC-P3, as well as an equimolar amount of PV1AGUA3 helper RNA. As shown

in Figure 4-5B, the 2C-P3 expressing template RNA supported nearly identical levels of

negative-strand synthesis as the replication deficient P3, and supported significantly lower levels

of negative-strand synthesis compared to those observed from the 2BC-P3 template RNA.

Taken together, these results strongly implicate cis translation of the 2BC-P3 precursor as the

primary requirement for efficient PV negative-strand synthesis. Multiple discrete proteins or

protein precursors derived from 2BC-P3 which include the 2B and 3Dpo1 polypeptides may

function synergistically to achieve efficient initiation of negative-strand synthesis. However,

given the obligatorily sequential nature of the PV polyprotein, the 2BC-P3 precursor would be

the minimum cis translation product required in either case for efficient negative-strand RNA

synthesis.

Discussion

The work presented here has clearly established a close coupling of PV translation and

initiation of negative-strand RNA synthesis in the in vitro HeLa S10 translation-replication









system, mirroring the characteristics of PV infection in vivo. Moreover, we have demonstrated

that this coupling is due to a marked cis preference of viral protein function, rather than the result

of RNA template preparation induced by ribosomal transit. Through the use of trans

complementation RNA replication assays in the cell-free system, we have defined this cis acting

protein product as the 2BC-P3 precursor polyprotein. This result, in combination with previous

observations by our lab and others, allows us to propose a model whereby the translation of the

2BC-P3 precursor in cis is followed rapidly by a concerted association with newly formed

membrane vesicles as well as its template RNA, initiating the critical process of replication

complex assembly. This model provides mechanistic insight into the functional coupling of PV

translation and initiation of RNA replication.

PV Translation in cis is a Prerequisite for Efficient RNA Replication

Using the HeLa S10 translation-replication reactions, we have observed that PV

subgenomic RNA which translates all its replication proteins in cis [P23 RNA] exhibits

approximately 5 to 10-fold higher levels of negative-strand RNA synthesis than the similar

frameshifted RNA [FS23 RNA], which obtains its replication proteins in trans. These

observations are consistent with the previous finding that all naturally occurring defective

interfering (DI) PV genomes maintain the translational reading frame, despite containing various

deletions in the P1 coding region (115). Additionally, previous work by our lab and others has

shown that maintaining the reading frame of PV RNAs through the majority of the P23 coding

sequence was required for efficient RNA replication in cell culture, even in the presence of a

helper RNA or helper virus (61, 151, 197). This phenomenon has also been observed in cell

culture during PV infection, in that genomes whose RNA replication has been arrested by

guanidine will, following release of the guanidine block, return to the ER to re-start translation

prior to RNA replication, despite the presence of sufficient PV proteins (70). In total, these data









strongly indicate that PV RNA replication requires not only newly translated replication proteins,

but that these nascent proteins be translated in cis from the PV genome which will subsequently

begin negative-strand RNA synthesis.

It is possible that the observed replication defect of PV RNAs resulting from prematurely

aborted translation is a secondary effect of decreased RNA stability. It is known that cellular

mRNAs which are improperly translated are subject to a wide range of RNA degradative

machinery, including nonsense mediated decay (56, 146). Recent work by Kempf and Barton

has also indicated that polyribosome assembly on PV RNA imparts some protection from

endogenous exoribonucleases, suggesting that the absence of polysomes would result in RNA

degradation (107). Interestingly, we observed no increase in the degradation of RNAs which

prematurely terminated translation. This observation is again consistent with previous

observations by our lab and others that premature termination of PV RNA does not result in

RNA instability (105, 151; Ogram et al., unpublished results). These results show that the RNA

replication defect observed in frameshifted PV RNAs is not a secondary effect of decreased

RNA stability, but instead is a direct result of the incomplete cis translation of the replicating

RNA.

Complete Ribosome Transit Through a Template RNA is Not Sufficient to Promote High
Levels of Negative-strand RNA Synthesis

Because initiation of negative-strand RNA synthesis and termination of translation both

occur at the extreme 3' end of the PV genome, it is possible that complete ribosomal transit and

translation termination in the authentic RNA context is required for preparing the 3' end of the

genome for efficient initiation of negative-strand synthesis. Using constructs which contain the

authentic translation termination context but do not translate any functional replication proteins,

we observed no significant difference between RNA which translated its entire open reading









frame (P1-3D*) compared to an RNA which terminated translation prematurely (FS1-3D*).

This clearly showed that neither complete ribosome transit, nor authentic translation termination,

conferred the ability to efficiently initiate negative-strand RNA synthesis.

A set of very elegant in vivo experiments was performed by Novak and Kirkegaard using

PV RNAs containing premature amber stop codons in which replication was assayed in both

non-permissive and amber-suppressor cells (151). Using this system, Novak and Kirkegaard

observed a severe replication defect in PV RNAs which prematurely terminated translation and

proposed a series of potential models to explain their results. One such model proposes that the

act of ribosomal transit through a critical region of the template RNA promotes efficient

negative-strand synthesis by affecting RNA secondary structure or by affecting protein

association with the RNA template. We observed that the cis translation of the P23-3CSTOP RNA

or 2C-P3 RNA did not result in efficient initiation of negative-strand synthesis, yet these two

RNAs together contain the entire RNA sequence which comprises the critical region identified

by Novak & Kirkegaard. This indicates that the efficient initiation of negative-strand RNA

synthesis observed on fully translated template RNAs is a result of the cis action of a viral

protein products) rather than the physical result of ribosomal transit through a specific region of

the PV RNA.

Poliovirus RNA Replication Requires Translation of the 2BC-P3 Precursor in cis

By providing all essential PV proteins and naturally occurring precursors in trans from a

helper RNA, we assayed for negative-strand synthesis from PV subgenomic RNA expressing

sequentially larger portions of the PV replication polyprotein. Using this additive approach, we

determined that the protein product required in cis for efficient negative-strand synthesis was

either 3DP"o or a 3D-containing precursor protein. By assaying for negative-strand synthesis

from RNAs expressing increasingly larger 3D-containing precursor proteins, we determined that









the 2BC-P3 precursor was the minimal PV polyprotein precursor required in cis to efficiently

initiate negative-strand synthesis. This concept is consistent with previous work which showed

that deletions extending past the 2Apro coding sequence are lethal in vivo, and that providing

2Apro in trans is sufficient to fully complement a 2Apro deficient PV replicon RNA (61, 104,

105). Under conditions where 2Apro is being provided in trans, such as those in Figure 4-4, the

addition of the 2A polypeptide to the cis precursor (i.e. comparing 2BC-P3 to P23) actually is

disadvantageous, forcing an additional cleavage step prior to generation of the ideal precursor.

This would explain the observation that 2BC-P3 RNA replicates better than P23 RNA when in

the presence of a helper RNA.

It remains possible that, rather than the 2BC-P3 precursor in its entirety, it is the function

of multiple distinct proteins or protein precursors within 2BC-P3 that are required in cis.

However, studies from the laboratory of Eckhard Wimmer showed that a dicistronic PV RNA

containing the EMCV IRES between the 2A and 2B coding sequence generated viable virus and

showed no abnormal polyprotein processing, whereas insertion of the EMCV IRES at any other

intergenic position in the replicase polyprotein was lethal (139, 160). These studies clearly show

that an intact 2BC-P3 precursor is essential for efficient PV RNA replication. Previous work

from our lab further establishes the critical nature of the 2BC-P3 precursor by showing that a

lethal mutation in protein 2C (2C[P131N]) could only be complemented efficiently by 2BC-P3

and not by a smaller precursor (104). It is critical to note, however, that despite the ability of the

2BC-P3 precursor to complement in trans, the ability of 2BC-P3 to promote negative-strand

RNA synthesis is still dramatically more efficient in cis than in trans. Taken together, these

observations indicate that the entire 2BC-P3 precursor as the essential cis acting viral protein

factor required for the efficient initiation of negative-strand RNA synthesis.









A Model for PV RNA Replication Complex Formation Dependent on cis Translation of the
2BC-P3 Precursor Polyprotein

Poliovirus replication occurs on membranous vesicles induced upon infection by a

combination of hydrophobic viral proteins and protein precursors. Interestingly, when these

vesicles are induced via heterologous expression of these proteins, they are not utilized for RNA

replication by a superinfecting virus (71). As the authors of this previous study concluded, this

indicates that membrane vesicles must be induced immediately prior to RNA replication, by the

proteins produced from the genome about to be replicated. This would require at least cis

translation of 2CATPase and/or 2BC, since it has been shown that characteristic membrane

rearrangements are induced by these two proteins (9, 20, 50). It is also of note that the PV

polyprotein is subject to two distinct processing cascades as described by Lawson & Semler, one

soluble pathway and one which is membrane associated (119). The membrane associated

processing pathway initiates with the creation of the 2BC-P3 precursor, which we have identified

here as the critical cis acting PV protein responsible for efficient initiation of negative-strand

RNA synthesis. Furthermore, given the significantly short half-life of the 2BC-P3 precursor

observed by Lawson & Semler, we assert that the trans acting capability of the 2BC-P3

precursor is severely restricted both by its inherently transient nature, as well as its membrane

association. Based on our results presented above as well as work performed by multiple other

laboratories, we propose a replication model whereby the PV polyprotein precursor 2BC-P3 acts

in cis to bind its genomic RNA and simultaneously induce and associate with membrane

vesicles, forming an active PV replication complex. This concerted process acts to functionally

couple viral translation, membrane vesicle induction, and RNA replication, and represents a

critical transition in the PV life-cycle from genomic translation to RNA replication.









A. AGUA3
PVI L PI P2 P3
AGUA3
HelpeU NA IVP4 VP2 VP3 VP1 2A 2B 2C 3A13B 3C 3D
Helper RNA
(A)80


P23 RNA 4 LfA2A 2B 2C |3A|3BI 3C I 3D h_

(A)80


FS23 RNA =tII

(A)80


B. PVIAGUA3 C. PVIAGUA3
S P23 FS23
Time(h): 0 1 2 4 0 1 2 4

SlNow tINtN
1 2 34 5 6 7 8
1 2

Figure 4-1. Translation of a PV RNA template is a prerequisite for efficient negative-strand
synthesis. A) Schematic ofpoliovirus RNAs used in these experiments. PV1AGUA3
helper RNA contains the entire PV RNA sequence with a mutation in the 3'NTR,
rendering it incapable of RNA replication. P23 RNA encodes all essential viral
replication proteins, and FS23 RNA contains a frameshift mutation in the 2A coding
region of P23 RNA and does not express any functional protein B) Replication of
P23 RNA and FS23 RNA was measured using PIRCs isolated from HeLa S10
reactions and RNA product was analyzed by CH3HgOH gel electrophoresis and
autoradiography as described in Chapter 2. Each transcript RNA contained two
non-viral 5' G residues which permits only negative-strand RNA synthesis.
Equimolar amounts of PV1AGUA3 RNA were included in each reaction to provide all
naturally occurring viral proteins in trans. C) The stability of uniformly radiolabeled
P23 RNA or FS23 RNA in HeLa S10 reactions was measured as described in Chapter
2. Aliquots of the reaction mixtures were removed after the indicated incubation time
and the full-length RNA remaining was analyzed by denaturing CH3HgOH gel
electrophoresis and autoradiography. As before, an equimolar amount of PV1AGUA3
RNA was included in each reaction to recapitulate replication reaction conditions.
Gels depicted in panels B & C were generated by Dr. Nidhi Sharma.


















A. B.
P1-3D* RNA PV1AGUA3



(A)80
FS1-3D* RNA

^ [_1 1 2

(A)80



Figure 4-2. Physical ribosome transit of a template RNA is not sufficient to promote efficient
initiation of negative-strand synthesis. A) Schematic of poliovirus RNAs used in this
experiment. P1-3D* RNA encodes a fusion protein between the P1 coding region
and a non-functional carboxy-terminal portion of 3D. During translation of Pl-3D*
ribosomes completely transit the length of the template and terminate translation in
the authentic RNA context. FS1-3D* RNA contains a frameshift mutation early in
the P1 coding region ofPl-3D* RNA and terminates translation prematurely, without
completely transiting the RNA template. B) Replication ofP1-3D* and FS1-3D were
assayed in the presence of equimolar amounts of PV1AGUA3 helper RNA using
PIRCs isolated from HeLa S10 reactions as described in Chapter 2. RNA product
was visualized by denaturing CH3HgOH gel electrophoresis and autoradiography.
The gel depicted in panel B was generated by Dr. Nidhi Sharma.









A.



Template RNA:


B.


2A 12B


I2C 3A 3B 3C 3D


P23-2CSTOP------- J

P23-3AST -----------j

P23-3BSTOP
P23-3CSTOP-- -

P23....------------------------.................................
P23----------------------------------.


(A)so


I1 2 3 4
1 2 3 4


Figure 4-3. Translation of 3D or a 3D precursor is required in cis for efficient initiation of negative-strand synthesis. A) Schematic of
poliovirus RNAs used in this study. Each successive template RNA encodes one additional protein component of the PV
replication polyprotein (P23), such that P23-2ASTOP encodes only 2A, P23-2BSTOP encodes 2AB, P23-2CSTOP encodes
2ABC, and so on. All RNAs used in these experiments are identical in length, and differ in sequence only by the inclusion
of two stop codons at the indicated position in the coding region. B) Replication of each template RNA indicated above
was assayed in the presence of equimolar amounts of PV1AGUA3 helper RNA as described previously. Full length
product RNA was analyzed by denaturing CH3HgOH gel electrophoresis and levels of negative-strand synthesis were
quantitated by phosphorimager. The level of negative-strand synthesis of each RNA were scaled relative to those observed
with P23 RNA and represented graphically below the autoradiograph. The gel depicted in panel B was produced by Dr.
Sushma Ogram.


P23-2ASTOP j

P23-2BSTOP


PV1AGUA3 Helper RNA

-' 0- 0 0
^v^ v^v^ v ^


5 6 7










A.
PVlp50 RNA



3D RNA


3CD RNA


3BCD RNA



P3 RNA


2BC-P3 RNA


P23 RNA


B.


T (A)80
A867-6011


3C 3D


h (A)80
4^Ulj~n(T)5n


(A)80

Bi 2C |3A 3C I 3D
(A)80

I 22 2C 3 3C (A 3D )
(A)80


PVIAGUA3 Helper RNA


> 4 4


1 2


3 4 5 11

3 4 5 6 7


140
" 120
100
so
80
| 60
S40
2 20
Z 0


Figure 4-4. Efficient initiation of negative-strand synthesis requires translation of 2B or a 2B precursor in cis. A) Schematic of
poliovirus RNAs used in this study. Each RNA encodes a successively larger 3DP"o precursor as indicated by the template
name listed at left. PVlp50 RNA acts as a control RNA, translating a non-functional 50 kDa protein and utilizing the
authentic translational start and stop contexts. B) Replication of each template RNA indicated above was assayed in the
presence of equimolar amounts of PV1AGUA3 helper RNA as described previously. Full length product RNA was
analyzed by denaturing CH3HgOH gel electrophoresis and quantitated by phosphorimager. The levels of negative-strand
synthesis of each RNA were scaled relative to P23 RNA and are represented graphically below the autoradiograph.













B.


PV1AGUA3


1 2 3


100
: 80
S60
2 40
20
0


Figure 4-5. Poliovirus RNA replication requires translation of the 2BC-P3 polyprotein precursor
in cis. A) Schematic of the poliovirus RNAs used in this study. P3 RNA encodes the
P3 polyprotein precursor, 2C-P3 RNA encodes the 2C-P3 precursor, and 2BC-P3
RNA encodes the 2BC-P3 precursor. B) Replication of each template RNA indicated
above was assayed in the presence of equimolar amounts of PV1AGUA3 helper RNA
as described previously. Full length product RNA was analyzed by denaturing
CH3HgOH gel electrophoresis and quantitated by phosphorimager. The levels of
negative-strand synthesis of each RNA were scaled relative to 2BC-P3 RNA and are
represented graphically below the autoradiograph.


A.


P3 RNA


FV81


4;;









CHAPTER 5
MUTLIPLE MOLECULES OF THE 3CD VIRAL PROTEIN PRECURSOR PERFORM
DISCRETE FUNCTIONS IN THE INITIATION OF POLIOVIRUS NEGATIVE-STRAND
RNA SYNTHESIS

Introduction

The differential use of polyprotein precursors and their products is a key strategy employed

by poliovirus (PV) to perform the many diverse functions required during viral replication using

limited sequence space. An extension of this is the evolution of multiple activities within a

single protein or protein precursor. The PV precursor 3CDpro exemplifies both of these concepts

in that it performs multiple functions as a precursor and these activities are functionally distinct

from is processed products 3Cpro and 3Dpro.

As a precursor, 3CDpro exhibits no polymerase activity, however its processed product

3Dp1', acts as the RNA-dependent RNA polymerase (RdRp) (73, 74, 88). The 3CDpro precursor

also has the ability to bind to stem-loop d of the 5'CL, and while this ability is partially retained

by its processed product 3Cpro, the binding affinity of 3Cpro for the 5'CL is 10-fold lower than

that of 3CDpro (12). And while both 3CDPro and 3Cpro are proteases, their cleavage specificities

and activity levels are different, and this difference is particularly apparent in the processing of

the viral capsid precursor (P1) and at the 3C-3D junction (157). In these cases, 3Cpro processing

of Pl and 3CD were 1000-fold and 100-fold less efficient than the processing observed by

3CDpro. Interestingly, there are very few structural differences between 3Cpro and 3DP"l alone

and within the 3CDpro precursor as determined by x-ray crystallography.(130).

The current model of PV replication complex formation invokes genomic circularization

mediated by RNP complexes formed at the 5'CL and 3'NTR/poly(A) tail to promote initiation of

negative-strand synthesis (26, 93, 126, 196). Given that 3CDPro, in the presence of PCBP and/or

3AB, was observed to form RNP complexes with the 5'CL as well as the 3'NTR (12, 14, 89,









158, 213), these circular models also included two molecules of 3CDpro. It has been established

that the formation of the 5'CL-3CDpro RNP complex is required for negative-strand synthesis

(12, 158, 213), but the functional role of 3CDpro bound to the 3'NTR is yet to be elucidated. The

consequences of differential interactions between 3CDPro and either 3AB or PCBP have also not

yet been addressed. Additionally, it has not yet been determined if the molecule binding to the

5'CL is the same as that which binds the 3'NTR or if these are indeed two different molecules as

has been modeled.

Although it has been established that the above described activities of 3CDPro are required

for PV RNA replication, the precise molecular mechanisms which drive these requirements have

not been delineated (12, 89, 158, 213). In an effort to more directly characterize some of these

mechanisms, we utilized the HeLa S10 translation-replication system to examine the

complementation profiles of functionally defined mutants in the PV protein precursor 3CDPro.

To do this, we performed trans complementation analysis of PV subgenomic RNA replicons

containing lesions in the 3CDPro coding sequence. These mutant RNAs were assayed for their

ability to assemble functional replication complexes and initiate negative-strand synthesis in the

presence of complementing protein expression RNAs. Herein, we demonstrate that 3Dpol must

be admitted into the replication complex as its immediate precursor 3CDpro and that binding to

the 5'CL is not required for this activity. In addition, we provide compelling evidence that at

least two molecules of 3CDpro are present in the PV replication complex and these individual

precursors perform multiple distinct functions. Lastly, we show that the 5'CL RNP complex

essential for initiation of negative-strand synthesis is likely formed by a molecule of 3CDPro

which enters the replication complex in the form of its precursor P3.









Results

Mutations Which Prevent the Production of Active 3Dpo" are Rescued by 3CDpro

To begin to analyze the role of 3CDpro in the formation of the PV replication complex used

to initiate negative-strand RNA synthesis, we first needed to establish the phenotypes of each

specific 3CDpro mutants in the HeLa S10 translation-replication system. The first subtype of

3CDpro mutants examined were those which failed to generate active 3Dpo1. The first of these

was a previously described mutant in the highly conserved YGDD motif of RNA-dependent

RNA polymerases, where the Gly327 of PV 3DP"o was mutated to Met (3D[G327M]) (98). This

mutation was shown to abolish all polymerase activity in bacterially expressed recombinant PV

3DPo1. The second mutant 3CDpro examined contained four sequential mutations of the 3C-3D

cleavage site, all on the 3Cpro side of the junction in positions P1-P4, thereby maintaining the

integrity of the 3DP"o amino acid sequence. This processing mutant (3CD[PM]) combines two

previously described processing site mutations [T181K, Q182D] with two additional mutations

[S183G, Q184N] designed to completely abrogate 3C-3D processing (12, 37, 88). This

extensive mutagenesis is required to completely inhibit processing of 3CDPro, as individual

mutations as well as combinations thereof have been shown to reduce, but not eliminate

processing ((88); data not shown). Because the 3CDpro precursor does not possess any of the

polymerase activity of its progeny 3DP"o, the abrogation of 3CD processing also functionally

inactivates polymerase activity (73, 74, 88). Since both 3CD[G327M] and 3CD[PM] would be

unable to generate a functional 3DP"0, PV RNAs containing these mutations should be unable to

replicate.

Each mutant 3CDPro was assayed for its ability to support negative-strand RNA synthesis

of a previously described subgenomic PV RNA replicon (P23 RNA) which contained the above

described mutations in the 3CD coding region. Negative-strand synthesis was assayed using









PIRCs isolated from HeLa S10 translation-replication reactions as described in Chapter 2.

Radiolabeled full-length product RNA was visualized by denaturing CH3HgOH gel

electrophoresis and autoradiography as previously described. As expected, P23 RNA which

expresses wild-type 3CDpro generated significant amounts of negative-strand product RNA

(Figure 5-1A, lane 1). However, P23 RNAs which express a 3CDpro that cannot generate active

3DP"o are unable to generate detectible levels of negative-strand synthesis (Figure 5-1A,

lanes 2-3).

To ensure that the observed RNA replication phenotype of these mutants was not the result

of defects in translation or processing, protein synthesis in the replication reactions was analyzed

by [35S]methionine incorporation, SDS-PAGE, and autoradiography. As shown in Figure 5-1B,

both mutant P23 RNAs generate similar levels of protein synthesis. Further, there are no

significant differences in the pattern of polyprotein processing, except where 3Cpr0 and 3DP"o

were absent from reactions expressing the 3CD[PM], as expected (Figure 5-1B, lane 3). These

data confirm that mutations which prevent the generation of active 3DP"o block PV

negative-strand RNA synthesis, and the mutations tested do not affect translation or polyprotein

processing.

To determine if these 3DP"o deficient mutations can be complemented in trans,

negative-strand synthesis of P23-3D[G327M] RNA or P23-3CD[PM] RNA was assayed in the

presence of non-replicating helper RNAs encoding sequentially larger 3D containing precursors.

Levels of negative-strand synthesis were assessed as described above. Interestingly, expression

of wild-type 3DP"o alone was not sufficient to rescue negative-strand synthesis to significant

levels for either of the 3DP"o deficient mutants (Figure 5-2A/2C, lane 1). However,









negative-strand synthesis of these mutants was efficiently restored by complementation with

3CDpro or a larger precursor (Figure 5-2A/2C, lane 2-4).

Protein synthesis and polyprotein processing in the replication reactions was also analyzed

by [35S]methionine incorporation, SDS-PAGE, and autoradiography. Despite the presence of a

disproportionate excess of polymerase in reactions which expressed solely 3DP"o (Figure 5B/5D,

lane 1), only minimal levels of negative-strand synthesis were observed in complementation

assays. Furthermore, there is direct correlation between the amounts of 3CDpro present and the

level of trans complementation observed in RNA replication assays. Taken together, these data

suggest that the active 3DP"' is delivered to the replication complex in the form of its immediate

precursor, 3CDPro.

Complementation of 3DP"o Deficient Mutations Requires the Intact 3CDpro Precursor

To further characterize the complementation of the 3DP"o deficient mutations by 3CDpr,

negative-strand synthesis of P23 RNAs expressing either 3CD[G327M] or 3CD[PM] was

assayed in the presence of a combination of 3Cpro and 3DP"o expression RNAs or an RNA which

expresses the heterologous mutant 3D/3CD. Levels of negative-strand synthesis were assessed

as described above. As before, complementation of both 3DP"o deficient mutants by 3CDpro was

significantly more efficient than complementation by 3DP"o (Figure 5-3A/3C, lanes 1-2).

Complementation using a combination of 3Cpro and 3DP"o expression RNAs was slightly less

efficient than using a 3DP"l expression RNA alone, and negative-strand synthesis was

undetectable in complementation reactions containing the heterologous 3D/3CD mutant

expression RNA (Figure 5-3A/3C, lanes 3-4). Analysis of protein synthesis and polyprotein

processing showed equal levels of protein synthesis and processing, except where expected for

additional proteins expressed in trans (Figure 5-3B/3D). These results clearly show that the









active 3DP1l used to initiate negative-strand RNA synthesis is delivered to the PV replication

complex in the form of its intact 3CDpro precursor.

Mutations Which Disrupt 3CPro/3CDpro Binding to the 5'CL Block RNA Replication and
Affect Polyprotein Processing

To further analyze the role of 3CDpro in the formation of the PV replication complex used

to initiate negative-strand RNA synthesis, we generated mutants in the RNA binding region of

3CPro/3CDpro and determined the replication and translation phenotypes of these mutants in the

HeLa S10 translation-replication system. Three distinct regions of the 3C primary sequence

have been implicated in binding to the 5'CL, an N-terminal region (Y6, K12, R13), a central

region (K82, F83, R84, D85, 186, R87), and a C-terminal region (T154, G155, K156) (12, 33, 36,

132, 142). The residues included in the C-terminal RNA binding region have also been

implicated VPg uridylylation on the cre(2C) hairpin and overlap a predicted protein-protein

interaction site (130), making this region unattractive for mutagenesis. The residues in the

central region represent a highly conserved picornaviral KFRDIR 3C-RNA binding motif, and a

previously described mutant, 3C[R84S], has been included in this analysis as a prototypic

example of mutations in this region(36). Lastly, a double mutant in the N-terminal RNA binding

region of 3Cpro was also created which combined two adjacent previously described RNA

binding mutations, 3C[K12N/R13N] (36).

Each 3CD RNA binding mutant (RBM) was assayed for its ability to support

negative-strand RNA synthesis of P23 RNA which contained one the above described mutations

in the 3CD coding region. The levels of negative-strand synthesis from isolated PIRCs were

assessed as described above. As shown previously, P23 RNA which expresses wild-type 3CDpro

generated significant amounts of negative-strand product RNA (Figure 5-4A, lane 1). However,









P23 RNAs which express a 3CPro/3CDpro that cannot bind the 5'CL are unable to generate

detectible levels of negative-strand synthesis (Figure 5-4A, lanes 2-3).

To determine the translational and processing phenotypes of these mutants, protein

synthesis in the replication reactions was analyzed by [35S]methionine incorporation,

SDS-PAGE, and autoradiography. As shown in Figure 5-4B, both mutant P23 RNAs generate

similar levels of protein synthesis, however, both mutants exhibited differences in the pattern of

polyprotein processing (Figure 5-1B, lane 2-3). In reactions containing P23-3C[R84S], there

was a significant accumulation of unprocessed high molecular weight precursors. Likewise,

every mutation tested within the conserved KFRDIR motif exhibited some degree of polyprotein

processing defect (data not shown), which complicates the interpretation of RNA replication

phenotypes. In reactions containing P23-3C[K12N/R13N], there was a moderate but detectable

increase in the efficiency of 3C-3D processing, however this is likely benign, particularly since

all other polyprotein processing seems unaffected. These data, particularly the 3C[K12N/R13N]

mutant, indicate that mutations which disrupt the binding of 3Cpro/3CDpro to the 5'CL block PV

negative-strand RNA synthesis

Complementation of 3Cpro/3CDpro RNA Binding Mutants Requires the Intact 3CDpro
Precursor

Although 3Cpro can bind RNA, it has been shown that the 3CDpro precursor has a 10-fold

higher affinity for the PV 5'CL than does 3Cpro alone (12). And given the significant excess of

3CDpro over 3Cpro that exists during PV infection, the most likely 5'CL RNP complex is one

which contains 3CDpro rather than 3Cpr alone. To test this assumption, complementation assays

were performed using P23 RNA containing either the 3C[K12N/R13N] or 3C[R84S] mutations,

in combination with non-replicating helper RNAs expressing either 3Cpr alone or its precursor









3CDpro. In addition, the mutant P23 RNAs were each complemented with a combination of 3Cpro

and 3DP"o expressing RNAs or an RNA which expressed the heterologus 3CDPro[RBM].

As predicted, the defect in negative-strand synthesis of both P23[RBM] RNAs were unable

to be complemented by 3Cpro alone or 3Cpro and 3DP"o in combination (Figure 5-5A, lanes

1, 3, 5, and 7). In contrast, expression of the 3CDpro precursor was able to complement both

P23-3C[K12N/R13N] as well as P23-3C[R84S], while the heterologous 3CD[RBM] was unable

to do so (Figure 5-5A, lanes 2, 4, 6, and 8). It is of note, however, that the efficiency of rescue

differed significantly between the two mutants, most likely as a result of interference by the

defective polyprotein processing exhibited by 3CD[R84S].

Analysis of protein synthesis in these reactions shows appropriate expression of all

complementing proteins as well as similar levels of protein synthesis for both P23 [RBM] RNAs

(Figure 5-5B/5C). Interestingly, some of the polyprotein processing defect exhibited by

P23-3C[R84S] is complemented in trans by 3Cpro/3CDPro, and this complementation is even

greater in the reaction which expressed 3CD[K12N/R13N] which exhibited elevated processing

activity (Figure 5-5C). Despite these minor processing irregularities, these results clearly show

that the intact 3CDpro precursor is required to complement a 3Cpro RNA binding mutation.

Moreover, this confirms that 3CDPro, and not 3Cpro, is a component of the 5'CL RNP complex

required for the initiation of negative-strand synthesis.

Complementation Between Two Functionally Distinct 3CDpro Mutants

Work by Cornell et al. showed reciprocal complementation between an RNA expressing a

non-functional chimeric polymerase and an RNA expressing a P3 precursor which contained a

3C/3CD RNA binding mutation (62). The authors therefore concluded that viral proteins

capable of binding RNA and initiating replication complex formation, can recruit complementing

proteins to the replication via protein-protein interactions. In the context of the current









investigation, this would predict that a 3DP"o deficient 3CDpro mutant which retained RNA

binding ability could be complemented by a 3CD[RBM] which could generate functional

polymerase. Replication complexes would therefore require the presence of at least two

molecules of 3CDpro for such a complementation to occur, and the potential replication complex

models for this are depicted in Figure 5-6.

To determine if reciprocal complementation of two functionally distinct 3CDpro mutants

was possible, negative-strand RNA synthesis was assayed in reactions containing

P23-3CD[G327M] RNA in combination with a helper RNA which expressed a wild-type or

mutant 3CDpro. For the purposes of these complementation experiments, 3CD[K12N/R13N] was

the RNA binding mutant of choice due to the severe processing defects exhibited by 3CD[R84S].

In reactions containing P23-3D[G327M], efficient complementation was observed in the

presence of wild-type or K12N/R13N 3CDpro RNA, but not in the presence of the synonymous

3CD[G327M] RNA (Figure 5-7A, lanes 1-3). When the complementation was reversed,

P23-3CD[K12N/R13N] was able to be complemented by both wild-type and G327M 3CDpro

RNA, but not by the synonymous 3CD[K12N/R13N] RNA (Figure 5-7A, lanes 4-6). However,

the reversed complementation efficiency was significantly reduced relative to the original

complementation, even in the presence of wild-type 3CDpro helper RNA.

To determine if the above complementation would also occur for the similarly 3DP"o

deficient 3CD[PM], negative-strand RNA synthesis was assayed in reactions containing

P23-3CD[PM] RNA in combination with a helper RNA which expressed a wild-type or mutant

3CDpro. As expected, efficient complementation was observed in reactions containing

P23-3D[G327M] RNA in the presence of wild-type or K12N/R13N 3CDpr' RNA, but not in the

presence of the synonymous 3CD[PM] RNA (Figure 5-7C, lanes 1-3). And here too, when the









complementation was reversed, the same pattern and decrease in efficiency of complementation

was observed as was for the 3CD[G327M] mutation (Figure 5-7C, lanes 4-6).

To determine if the differences in complementation efficiencies were caused by

abnormalities in translation and/or polyprotein processing, protein synthesis was monitored by

[35S]methionine incorporation and assessed by SDS-PAGE and autoradiography as before. As

shown in Figures 5-7B and 5-7D, overall protein synthesis was nearly identical in all reactions

and polyprotein processing showed no abnormalities (except for expected differences for

3CD[K12N/R13N] and 3CD[PM] as previously discussed).

These results clearly show that two distinct mutations in essential functions of the 3CDpro

precursor can be reciprocally complemented to restore replication complex formation and

negative-strand synthesis. Therefore, two or more molecules of the 3CDpro precursor must be

simultaneously present in the PV replication complex, such as diagrammed in Figure 5-6D.

High Efficiency Complementation of 3C[K12N/R13N] Requires the P3 Precursor

Interestingly, although the same pattern of complementation was present, we observed a

significant decrease in complementation efficiency when the template RNA contained the

3CD[K12N/R13N] mutations. This could result from either a dominant negative effect of a

larger 3CD[K12N/R13N] containing precursor, a more stringent requirement for proteins in cis

to bind the 5'CL, or the requirement of a larger precursor to provide RNA binding in trans. To

test this, we first assessed the ability ofP23-3C[K12N/R13N] RNA to be complemented by

RNAs expressing sequentially larger 3Cpr0 containing precursors. As before, in reactions

containing P23-3C[K12N/R13N] RNA and 3Cpro expression RNA, levels of negative-strand

synthesis were undetectable, whereas complementation was observed in the presence of RNA

which expressed 3CDpro (Figure 5-8A, lanes 1-2). Surprisingly, higher levels of negative-strand

synthesis were observed when P23-3C[K12N/R13N] RNA was complemented with P3 or









2BC-P3 expression RNA (Figure 5-8A, lanes 3-4). Aside from the expected increases in the

levels of additional viral proteins expressed in trans, there were no significant alterations in the

translation or polyprotein processing profile in any of the replication reactions (Figure 5-8B).

These results clearly show that high efficiency complementation of a 3CDPro RNA binding

mutant requires the presence of a P3 precursor. Moreover, these data suggest that the P3

precursor delivers 3CDPro to the 5'CL during the formation of the 5'CL RNP complex which is

essential for the initiation of negative-strand RNA synthesis.

Discussion

The work presented here has clearly illustrated the multifunctional nature of the viral

3CDpro precursor, particularly as it pertains to the initiation of negative-strand RNA synthesis.

By performing trans complementation assays using the HeLa S10 translation-replication system,

we have further defined the role of 3CDPro, as well as its precursors and processed products, in

the formation of a functional PV replication complex. Using this approach, we have clearly

shown that 3DPo1 is admitted to the replication complex in the form of its intact immediate

precursor 3CDpro and that binding to the 5'CL is not a prerequisite for this activity. Furthermore,

by performing reciprocal complementation using two 3CDPro mutants in distinct, essential

functions, we have shown that there are at least two molecules of 3CDpro present in the PV

replication complex which perform discrete functions. Lastly, we have shown that the 3CDPro

which forms the essential 5'CL RNP complex is likely admitted to the replication complex in the

form of its precursor P3.

Active 3DP"o is Admitted to the PV Replication Complex in the Form of its
Polymerase-inactive Precursor 3CDpro

Although 3CDpro contains the entire 3Dpo1 peptide, it contains none of the associated

polymerase activity (73, 74, 88). This is most likely due to changes in positioning of the









N-terminus of 3Dp1l that occur subsequent to processing (95, 130, 180, 199). This strategy

allows PV to synthesize large amounts of 3CDpro prior to replication without risking the

generation of non-specifc dsRNA products on cellular mRNAs which could activate innate

immune pathways. Here, the 3CDpro precursor functions as a pro-enzyme which can be

synthesized to high-levels and activated rapidly on demand. We were able to show that mutants

in either 3C-3D processing or the conserved 3DP"o RdRp motif could only be rescued efficiently

by an intact 3CDpro or a 3CD containing precursor. This indicates that 3CDpro is recruited into

the PV replication complex in an inactive form, and its activation by processing represents the

"firing" of replication complexes and marks the initiation of negative-strand synthesis.

Additionally, this recruitment does not require direct binding of the 3CDpro precursor to RNA,

since mutations which disrupted conserved RNA binding residues in 3C were able to

complement 3DP"o deficient RNA replicons.

RNA Binding and Protease Activities of 3CDpro are Functionally Linked

Each RNA binding mutation tested, in addition to its replication phenotype, also exhibited

altered patterns of polyprotein processing. In most cases, this alteration was detrimental and

resulted in accumulation of unprocessed precursors, however in one case (3C[K12N/R13N]) the

mutations resulted in increased processing efficiency. The effect of the latter mutations was mild

and manifested primarily as an increased proportion of processed 3Cpr0 and 3DP"o in replication

reactions. These observations are consistent with recent structural work by Claridge et al., who

showed that RNA binding by rhinovirus 3Cpro induced conformational changes in regions

involved in proteolysis (53). In this manner, one face of 3Cpro/3CDpro communicates with the

other to transmit information regarding RNA binding status to the proteolytic machinery. This

has significant implications for the PV life-cycle, since the rapid polyprotein processing that is

observed in the membrane associated processing cascade may actually be performed by the









5'CL-3CDpro RNP complex. Since this processing pathway is associated with RNA replication,

polyprotein processing, membrane association, and replication complex formation may be

additionally coupled by enhanced proteolysis by RNA-associated 3Cpro/3CDpro. By this model,

most RNA binding mutations which disrupt processing may essentially lock 3Cpro/3CDpro in an

unbound conformation, whereas the K12N/R13N mutant induces conformational shifts that

simulate the bound conformation in the absence of RNA.

Multiple 3CDpro Peptides are Present in the PV RNA Replication Complex Used to Initiate
Negative-strand RNA Synthesis

Current models of initiation of PV negative-strand RNA synthesis involve interaction of

the 5' and 3' ends of genomic RNA, mediated by RNP complexes, to form a circular replication

complex (26, 93, 126, 196). It was known that 3CDPro, in the presence of PCBP and/or 3AB,

could form RNP complexes with both the 5'CL and the 3'NTR (12, 14, 89, 158, 213). Based on

this, in combination with our own 3D complementation data, our model for circular replication

complex formation included two molecules of 3CDpro. Later studies by Cornell et al. showed

that negative-strand synthesis of an RNA encoding an inactive chimeric 3DP"o could be

complemented by expressing a P3 precursor with an RNA binding mutation (62). However, the

authors did not characterize in which function the chimeric polymerase was defective and

examined only the P3 precursor for its ability to complement in trans. From this data it is

difficult to draw precise conclusions about replication complex formation and composition.

Using precise mutations which inactivated single functions of the 3CDpro precursor, we

demonstrated reciprocal complementation of 3Dpol deficient mutants (3CD[G327M] and

3CD[PM]) with an RNA binding mutant (3CD[K12N/R13N]). Each of the mutants blocks

negative-strand RNA synthesis as each represents a mutation(s) in a discrete but essential

function of the PV replication complex. Since both functions are required simultaneously in the









initiation of negative-strand synthesis, complementation of these mutants requires that at least

one copy of each mutant 3CDPro be present at the time of replication initiation. This represents

the first conclusive functional evidence that multiple molecules of the 3CDpro polypeptide are

present and perform discrete functions within the PV replication complex that is used to initiate

negative-strand RNA synthesis.


The 3CDpro Bound to the 5'CL is Admitted to the PV Replication Complex in the Form of
its Precursor P3

We observed that, although functional, RNAs expressing 3CDPpr, 3CD[G327M], or

3CD[PM] were only capable of minimally complementing P23-3CD[K12N/R13N] RNA. This

was significant because when the complementation had been reversed, 3CD[K12N/R13N]

expression RNA was capable of complementing P23-3D[G327M] and P23-3CD[PM] RNAs to

significantly higher levels. Upon examining the ability of larger 3Cpr0 precursors to rescue

negative-strand synthesis of P23-3C[K12N/R13N] RNA, we showed that complementation

efficiency was significantly higher in the presence of either P3 or 2BC-P3 expression RNAs.

Given that expression of P3 resulted in the highest level of negative-strand synthesis, and that

expression of 2BC-P3 also provides P3, we conclude that the 3CDpro which forms the essential

RNP complex with the 5'CL is first admitted into the replication complex in the form of the P3

precursor. This is particularly interesting, since 3CDpro has been shown to bind to the 5'CL in

the presence of the 3AB precursor (89, 213). Together, 3AB and 3CDpro comprise the P3

precursor, which may enter the replication complex intact and subsequently process upon

binding to the 5'CL. Furthermore, since VPg(3B) serves as protein primer for RNA synthesis,

the 3AB generated from above described P3 processing, could serve as the precursor for the VPg

used to prime negative-strand synthesis. This model is consistent with previous work which









showed that mutations in VPg which blocked its priming ability could only be complemented in

trans by P3 or in cis as an intragenic fusion to a 3CDprocontaining precursor (124).
























6- 3CD-
I U
A B B-







_3C-










3A2BC-
3A-
1 2 3








123

Figure 5-1. Mutations which prevent the generation of active 3DP"o block RNA replication.
A) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10
reactions as described in Chapter 2. Reactions contained subgenomic P23 RNA
containing either a wild-type or mutant 3CD coding region (3D[G327M] or
3CD[PM]). Full length RNA product was analyzed by denaturing CH3HgOH gel
electrophoresis and autoradiography. B) A portion of the HeLa S10 reactions
described in (A) was metabolically labeled with [35S]methionine to assay for protein
synthesis. These reactions were analyzed by SDS-PAGE and autoradiography. Viral
proteins are indicated at left.











A.


SU
ff)


C.


(-
1')P
ELM Cl


P23 3D[G327M]





1 2 3 4


B.



P23 3DIG327M]



3D-

2BC- IM'IS *I*'


2C-




3C-

2A- at .
3AB-
3A-


1 2 3 4


n Q


P23- 3CDIPM]


D.


1 2 3 4






P23 3CDIPM1


3CD-

3D-


eA- ---^
2c-




3C-

2A-
3AB-
3A-


1 2 3 4


Figure 5-2. Viral Precursor 3CDpro complements both 3D[G327M] and 3CD[PM] in trans.
A & C) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10
reactions as described in Chapter 2. Reactions contained P23 RNA with a mutated
3CD coding region (3D[G327M] or 3CD[PM]) and a second complementing RNA
expressing the indicated protein. All complementing RNAs contain the AGUA3
mutation which inhibits negative-strand synthesis. Product RNA was analyzed by
denaturing CH3HgOH gel electrophoresis and autoradiography. B & D) A portion of
the reactions described above was metabolically labeled with [35S]methionine and
these reactions were analyzed by SDS-PAGE and autoradiography. Viral proteins are
indicated at left.


iff
~lc~)
cC, 01
Pill (V















(Es
ra r'


Q |
cr &<
+ I
u U
n n


U
C' u


P23 3D[G327M]


N
r-

+ 0
+ Q
U U
n n


P23- 3CDIPM]


U


1 2 3 4





S + I


P23 3DIG327MI


1 2 3 4



P tD
C + I


P23 3CDIPMI


3CD-

3D-
2BC-


2C- OReI*



3C- -

2A- -
3AB-
3A-


3CD- ig;3

3D-







3C--
2BC-








2A- ,-R e,

3AB-
3A-


1 2 3 4 1 2 3 4
Figure 5-3. Complementation of 3D[G327M] or 3CD[PM] requires the intact 3CDpro precursor.
A & C) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10
reactions as described in Chapter 2. Reactions contained P23 RNA with a mutated
3CD coding region (3D[G327M] or 3CD[PM]) and a second complementing RNA
expressing the indicated protein(s). All complementing RNAs contain the AGUA3
mutation which inhibits negative-strand synthesis. Product RNA was analyzed by
denaturing CH3HgOH gel electrophoresis and autoradiography. B & D) A portion of
the reactions described above was metabolically labeled with [35S]methionine and
these reactions were analyzed by SDS-PAGE and autoradiography. Viral proteins are
indicated at left.
















A. B.




ee
I I






Z3CD



2BC-


if- OW &C


1 2 3
3C-





3A-- -' 0
123

Figure 5-4. Mutations which disrupt 3CPro/3CDpr' binding to the 5'CL block RNA replication.
A) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10
reactions as described in Chapter 2. Reactions contained subgenomic P23 RNA
containing either a wild-type or mutant 3C coding region (3C[K12N/R13N] or
3C[R84S]). RNA product was analyzed by denaturing CH3HgOH gel electrophoresis
and autoradiography. B) A portion of these reactions described was metabolically
labeled with [35S]methionine to assay for protein synthesis. These reactions were
analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left.













+ Q + Q
U U U U C U U U
rl ) t f rC rn fnC
P23 3CIK12N/R13N] P23- 3CIR84S]


3 4 5 6 7 8


ase
0 +

P23-3CIK12N/R13NI





3D-

2BC- all e

2C-



3C-- -


C.
Z
@1

+

P23-3CIR84S1


3D-

a e
3Dc- .WrJ~


2C-
dua amf


3C-0


2A-- _.2A- -
3AB- "-
3A3A-
3A- 3A-
1 2 3 4 1 2 3 4
Figure 5-5. Complementation of 3C[K12N/R13N] or 3C[R84S] requires the intact 3CDpro
precursor. A) Negative-strand synthesis was assayed using PIRCs isolated from
HeLa S10 reactions as described in Chapter 2. Reactions contained subgenomic P23
RNA containing a mutant 3C coding region (3C[K12N/R13N] or 3C[R84S]) and a
second complementing RNA expressing the indicated protein(s). RNA product was
analyzed by CH3HgOH gel electrophoresis and autoradiography. B-C) A portion of
the above reactions was metabolically labeled with [35S]methionine to assay for
protein synthesis. These reactions were analyzed by SDS-PAGE and
autoradiography. Viral proteins are indicated at left.


I
1 2










B.


Cleavage of 3AB
and 3CD[G327M]

"


Cannot process to form 3D[


C.


3D[G327M]
Is catalytically inactive


3CD-RBM
Cannot bind to the 5'CL


1) Cleavage of 3AB and 3CD-RBM
2) Initiation of negative-strand sN nihesis


.-. .,. :!9


^2IIID
T* .
,r. [ T ,1


Figure 5-6. Schematic of trans complementation using two functionally distinct mutations in
3CDpro. A) In the presence of only 3CD[G327M], replication complexes could form
and process, however, the 3DP"o generated is catalytically inactive and RNA
replication is blocked. B) In the presence of processing mutant 3CDpro (3CD[PM]),
replication complexes could form, however, 3CD has no polymerase activity before it
is processed. Since 3CD[PM] cannot process, negative-strand synthesis is blocked.
C) In the presence of RNA binding mutant 3CDprO (3CD[RBM]), the essential RNP
complex at the 5'CL cannot be formed, and as a result, negative-strand synthesis is
blocked. D) If a 3CD[RBM] is co-expressed with either 3CD[G327M] or 3CD[PM],
the polymerase deficient precursor could bind the 5'CL and the RBM could provide
the polymerase. This would allow initiation of negative-strand synthesis.


A.


D.













3CD: G3
P23: G327M


It


2A-
3AB-
3A-


M e l


Z Z


3CD: P K | NR3
P23: PM K12N/RI3N


5 6


w


2 3 4 5 6


Z Z

Z Z
3CD: P I2R1
P23: PM K12N/R13N


3CD-

3D- -g
2BC-"ii;'

2c- ,33333


z z











3D- i
P- rZ FI









3C-D:
P23: G327M K12N/R13N







3D- Ap.1 f-








3C-


lo


2A- eia 4 ne ia
3AB-
3A-


1 2 3 4 5 6 1 2 3 4 5 6
Figure 5-7. Two functionally distinct 3CDpro mutants can complement each other in trans.
A & C) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10
reactions as described in Chapter 2. Reactions contained P23 RNA with a mutated
3CD coding region (3D[G327M]/3CD[PM] or 3C[K12NR13N]) and a second
complementing RNA expressing the indicated protein. All complementing RNAs
contain the AGUA3 mutation which inhibits negative-strand synthesis. Product RNA
was analyzed by CH3HgOH gel electrophoresis and autoradiography. B & D) A
portion of the reactions described above was labeled with [35S]methionine and
analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left.


N


K12N/RI3N


2 3 4


3C-









A


P23-3ClK12N/R13N]





P3-
P23 3CIK12N/R13N] 3CD-



2A- ... > |
3 -
U U 6- 4090












2BC-1234
1 2 3 4 2c-
3C1 40











1 2 3 4
Figure 5-8. Complementation of a 3CDpro RNA binding mutant is more efficient when P3 is
provided in trans. A) Negative-strand synthesis was assayed using PIRCs isolated
from HeLa S10 reactions as described in Chapter 2. Reactions contained
P23[K12N/R13N] RNA and a second complementing RNA expressing 3C or 3C
precursors of increasing size. All complementing RNAs contain the AGUA3
mutation which inhibits negative-strand synthesis. Product RNA was analyzed by
denaturing CH3HgOH gel electrophoresis and autoradiography. B) A portion of the
reactions described above was labeled with [35S]methionine and these reactions were
analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left


D









CHAPTER 6
SUMMARY AND CONCLUSIONS

In this dissertation, I have presented and discussed the results from three distinct yet

interconnected lines of investigation into the protein requirements for the initiation of poliovirus

negative-strand RNA synthesis. Each of these studies has generated significant insight into how

these key viral and cellular proteins function in PV replication complex assembly, and also to the

broader understanding of the replication of related enteroviruses. Techniques developed to

perform this work have already been applied to the study of other stages of the viral life cycle,

including PV translation and cre(2C)-dependent VPg uridylylation, and will soon be adapted for

characterization of Coxsackievirus B3 replication. Future work based on each of these lines of

investigation will provide a more detailed understanding of the molecular mechanisms by which

poliovirus, as well as other enteroviruses, regulate the critical steps of viral RNA replication.

The Role of PCBP in the Initiation of Poliovirus Negative-strand Synthesis

The first of the investigations presented herein probes the involvement of the

multifunctional cellular protein PCBP in virus replication. To do so, our laboratory has

developed and applied a novel protein-RNA tethering system to study of virus replication. Using

this system we were able to confirm the activity of PCBP in supporting negative-strand synthesis

and were further able to identify the functional domains within PCBP2. Morover, we were able

to show that some, but not all members of the PCBP protein family can function in PV RNA

replication. In future studies, these evolutionarily related, but functionally distinct isoforms can

be used to direct more detailed mutagenic studies of the individual functional domains. This

approach, in combination with the (MS2)2 protein-RNA tethering system, can then be used to

more precisely define the protein-protein interaction surfaces and binding partner critical to

PCBP's ability to promote negative-strand synthesis.









The (MS2)2 Protein-RNA Tethering System: Virus-Host Interaction

A defining characteristic of a virus is its ability to commandeer its host cell and subvert the

cellular machinery for its own replication. The (MS2)2 protein-RNA tethering system used in

this study provides an ideal framework for additional studies on virus-host interactions critical to

the understanding of virus replication and cellular protein functions therein. The specific

integration of key host proteins into defined steps in the viral life cycle relieves the need of the

viral genome to encode such proteins, but also functions as a post-entry determinant in cell

tropism. For viruses like poliovirus which infect multiple distinct cell types, these cellular

protein determinants could function as replicative rheostats, allowing the virus to tailor its

replication to the cell type it has infected. These unique interactions between viral and cellular

proteins are also very attractive antiviral drug targets, particularly given that cellular protein

evolution is not subject to the same selective pressures as viral proteins.

The (MS2)2 Protein-RNA Tethering System: Host Protein Function

An appealing extension to the (MS2)2 protein-RNA tethering system, in addition to the

generalized study of host protein involvement in the replication of other RNA viruses, is the

potential to better understand the normal cellular role of these key proteins. Viral systems serve

as microcosms for complex host cell processes, and have provided the foundation for much of

our current understanding of cellular biology. DNA replication, mRNA splicing, innate

immunity, endocytosis, oncogenesis, and apoptosis are among the many cellular processes

initially characterized using viruses or virus-based approaches. Likewise, by understanding

precisely how key cellular proteins are exploited during virus infection, we can better understand

their role in cellular processes and in the global regulatory networks in which they often

participate. This work would also extend to the role of these critical proteins in disease states,

some of which may be directly related to virus infection. Dysregulation of PCBP regulated









mRNAs has been linked to liver cirrhosis, cervical cancer, and cardiomyopathy (123, 169, 191,

198). Interestingly, each of these conditions can also result from infection by a virus that utilizes

PCBP during its replication: Hepatitis C Virus (HCV), Human Papillomavirus (HPV), and

Coxsackievirus B (CVB), respectively. These all serve as examples of complex disease states

where critical protein-protein and/or protein-RNA interactions could be initially examined in a

simplified context, using a virus or virus-based system in combination with the (MS2)2 tethered

function system.

The Role of Viral Protein Precursors in the Initiation of PV Negative-strand Synthesis

Modeling Formation of the PV RNA Replication Complex

The second and third lines of investigation both deal with the critical role of distinct viral

polyprotein precursors in the initiation of poliovirus negative-strand RNA synthesis. Firstly, we

examine the molecular basis for the requirement of genomic translation in cis to promote

efficient initiation of negative-strand synthesis. Using trans complementation assays, we

showed that the activity of a protein precursor, rather than physical ribosome transit, was

responsible for the observed cis enhancement of negative-strand synthesis. Further, we

identified the critical cis-acting precursor as 2BC-P3 and generated a model of replication

complex formation which accounts for this requirement. This model is able to account for the

previously observed coupling between genomic translation and RNA replication observed in

infected/transfected cells, as well as other previously reported protein complementation studies

from our lab.

The last line of investigation also utilized trans complementation assays in combination

with functionally defined mutants of the multifunctional viral precursor 3CDPro. Using this

approach, we defined the functional polyprotein precursor of the active polymerase in the

replication complex to be 3CDpro, however we showed that the preferred precursor utilized to









form the essential 5'CL RNP complex was P3. These experiments also validated the current

model for initiation of negative-strand synthesis, which depicts two functionally discrete 3CDpro

polypeptides within the PV replication complex. This study allowed us to enrich our model of

PV replication complex formation to include greater detail as to the source of protein precursors

which form the critical 5'CL RNP complex, provide the VPg primer for RNA synthesis, and

generate active 3DP"1.

Close Coupling of the Viral Life-Cycle Ensures Viral Fitness

Both of these studies, in addition to defining critical components of the PV replication

complex, also illustrate the tightly coupled nature of viral replication. In most cases,

complementation in trans of a viral protein supports significantly lower levels of negative-strand

RNA synthesis than would be observed if that protein was provided in cis. The evolutionary

imperative to tightly couple the different stages of the viral life-cycle stems from the complexity

inherent in coordinating a very intricate sequence of events in the context of the chaotic milieu of

a host cell, in the face of extensive innate anti-viral defenses. This task is only complicated

further by the high mutation rates exhibited by RNA viruses and the need to counter-balance the

increased speed of viral evolution with extensive genomic quality control. However, by doing

this, a virus ensures replication of complete genomes encoding fully functional proteins to the

exclusion of incomplete or defective genomes, preventing the wasteful use of valuable cellular

resources. Poliovirus, like other small RNA viruses, maximizes protein function using limited

genomic sequence space by encoding a single large polyprotein and utilizing each unique

precursor within the protein processing cascade. It now also appears that polyprotein processing

also contains within it the intrinsic ability to tightly couple cis translation of PV RNA and

subsequent replication complex formation. This coupling functions as a critical replication

checkpoint, a penultimate guarantee that the PV template RNA about to be replicated encodes a









functional set of essential replication proteins, ensuring efficient RNA replication and

evolutionary maintenance of viral fitness.









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BIOGRAPHICAL SKETCH

Allyn Russell Spear was born in Milwaukee, Wisconsin in August of 1981 to Neal and

Marlyn Spear. He grew up in Wauwatosa, Wisconsin and graduated from Wauwatosa East High

School in June 1999. Following this, Allyn attended the University of Wisconsin-La Crosse, and

graduated in May 2003 with bachelor's degrees in both microbiology and chemistry with an

ACS certification. While at the University of Wisconsin-La Crosse, Allyn had the opportunity to

train under the direction of Dr. Michael A. Hoffman, performing research on the role of the

matrix protein in the assembly and budding of human parainfluenzavirus type-3. In August

2003, Allyn began the Interdisciplinary Program in Biomedical Sciences at the University of

Florida. In the Spring of 2004, he had the opportunity to begin doctoral research in the

laboratory of Dr. James Bert Flanegan, studying the biochemistry and molecular biology of

poliovirus replication. Under Dr. Flanegan's direction, Allyn completed all required coursework

and dissertation research in the Summer of 2009.





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1 PROTEIN REQUIREMENTS FOR TH E INITIATION OF POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS By ALLYN R. SPEAR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Allyn R. Spear

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3 To my loving and supportive parents, who have always taught me to never stop working towards my dreams

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4 ACKNOWLEDGMENTS I would like to thank Dr. J. Bert Flanegan for the m any opportunities he has provided as well as the extensive scientific freedom he has granted me to pursue many varied avenues of research. I would like to thank the members of the Flanegan lab for their advice, support, assistance, and guidance. Brian ODonnell, Jo an Morasco, Nidhi Sharma, Sushma Ogram, and Jessica Parilla have all contributed in innumerable ways to my scientific development and have been and always will be my friends. And for the many insightful discussions and suggestions, I would like to thank all of the members of my co mmittee, Dr. Rich Condit, Dr. David Bloom, Dr. Linda Bloom, and Dr. Jorg Bungert. I would al so like to thank Dr. Rob McKenna for always being available and willing to listen; you have been like a second mentor to me. I would like to sincerely thank my parents for their undying support for almost every crazy thing I have wanted to try in my life, including crossing the country for graduate school. I would also like to thank my first scientific mentor, Dr. Michael Hoffman. His patience with me as a young scientist and his extensive mentoring provided me with the foundations of my scientific training. Dr. Hoffmans commitm ent to balancing his professional and personal life, and his dedication to scientif ic education, continues to be an inspiration to me to this day. Particularly for her exceptional patience and support during the writ ing of my thesis, I would like to thank Zenia Torres. She has given me a brighter outlook on my future than I ever could have dreamed and I cant imagine my life without her. Last, but certainly not least, I would like to more directly thank Sushma Ogram for her camaraderie and support throughout my graduate car eer. Selfless, caring, and a friend without condition, Sushma always helped bring me up duri ng the worst of times. I know that I would not have made it through graduate school without her, and for that I offer my sincerest thanks.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF FIGURES.........................................................................................................................8LIST OF ABBREVIATIONS........................................................................................................ 10ABSTRACT...................................................................................................................................11 CHAP TER 1 BACKGROUND AND SIGNIFICANCE ..............................................................................13Poliovirus Pathogenesi s and Epidemiology............................................................................ 13Poliovirus Molecular Biology................................................................................................. 15Attachment and Entry...................................................................................................... 16Viral Translation and Polyprotein Processing................................................................. 17Virus-Induced Alteration of Host Cell Environment......................................................18Host Protein Involvement in RNA Replication...............................................................19Negative-Strand RNA Synthesis.....................................................................................20Positive-Strand RNA Synthesis.......................................................................................21Packaging and Release of Progeny Virions..................................................................... 23Cell-Free Replication System................................................................................................. 242 MATERIALS AND METHODS........................................................................................... 28DNA Manipulation and Cloning Techniques.........................................................................28Site-Directed Mutagenesis...............................................................................................29Two-Step PCR.................................................................................................................30Construct Verification and DNA Stock Preparation.......................................................31cDNA Clones Used in These Studies.....................................................................................32Poliovirus Clones Used in These Studies........................................................................ 32Poliovirus-Based Protein Expressi on Clones Used in These Studies............................. 37Bacterial Protein Expression Cl ones Used in These Studies........................................... 41RNA Transcript Preparation and Purification........................................................................42Standard Transcription.................................................................................................... 42Ribozyme Optimized Transcription................................................................................ 435 Capping Transcription.................................................................................................43HeLa Extract Preparation.......................................................................................................43S10 Preparation...............................................................................................................44IF Preparation..................................................................................................................45HeLa S10 Translation-RNA Replication Reactions............................................................... 46RNA Programming and Translation................................................................................46PIRC Isolation and RNA Replication..............................................................................47Analysis of Protein Synthesis by SDS-PAGE........................................................................48

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6 Analysis of RNA Replication by Denaturing CH3HgOH Gel Electrophoresis...................... 48Bacterial Protein Expression................................................................................................... 49Electrophoretic Mobility Shift Assays.................................................................................... 50Riboprobe Synthesis........................................................................................................ 50Binding Reactions and Gel Electrophoresis.................................................................... 513 POLY(C) BINDING PROTEIN IS REQUIRED FOR EFFICIENT INITIATION OF POLIOVIRUS NEGATIVE-ST RAND RNA SYNTHESIS .................................................. 52Introduction................................................................................................................... ..........52Results.....................................................................................................................................55A Mutation in Stem-loop b of the 5 Cloverl eaf Inhibits Negative-strand Synthesis.....55(MS2)2 Protein-RNA Tethering System.......................................................................... 56(MS2)2PCBP2 Binds Specifically to 5CLMS2 RNA.......................................................57(MS2)2PCBP2 Restores Negative-strand Synthesis on P23-5CLMS2 RNA...................58Deletion Analysis of PCBP2 Using the (MS2)2 Protein-RNA Tethering System..........58The Conserved KH3 Domain is Sufficient to Support Negative-strand Synthesis.........60The Combined KH1-KH2 Domain Fragment Does Not Utilize PCBP Dimerization to Promote Negative-strand Synthesis......................................................................... 61Multiple PCBP Isoforms Support Initiation of Negative-strand RNA Synthesis........... 62Not All PCBP Family Members Support Negative-strand synthesis.............................. 64Discussion...............................................................................................................................65Prior Indications of PCBP Involvement in Poliovirus RNA Replication........................66(MS2)2 Protein-RNA Tethering Assay Demons trated that PCBP is Required for Poliovirus Negative-strand Synthesis.......................................................................... 67The Combined KH1 & KH2 Fragment or the KH3 Domain of PCBP2 is Required for Negative-strand Initiation....................................................................................... 68A Subset of PCBP Isoforms Support Init iation of Negative-strand RNA Synthesis......694 2BC-P3 IS THE CRITICAL CIS-ACTING VIR AL PROTEIN PRECURSOR DIRECTING INITIATION OF POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS...................................................................................................................... .....85Introduction................................................................................................................... ..........85Results.....................................................................................................................................86Efficient PV Negative-strand Synthesis Re quires Translation of Viral Template RNA.............................................................................................................................87Template RNA Translation Alone is No t Sufficient to Promote Efficient PV Negative-strand RNA Synthesis..................................................................................89Translation of the 3D Coding Region in cis is Necessary for Efficient PV Negative-strand Synthesis............................................................................................90Translation of the 2BCP 3 Protein Precursor in cis is Sufficient for Efficient PV Negative-strand Synthesis............................................................................................91Discussion...............................................................................................................................92PV Translation in cis is a Prerequisite for Ef ficient RNA Replication........................... 93Complete Ribosome Transit Through a Temp late RNA is Not Sufficient to Promote High Levels of Negative-strand RNA Synthesis.........................................................94

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7 Poliovirus RNA Replication Requires Tran slation of the 2BC-P3 Precursor in ci s .......95A Model for PV RNA Replication Complex Formation Dependent on cis Translation of the 2BC-P3 Precursor Polyprotein.......................................................975 MUTLIPLE MOLECULES OF THE 3C D VIR AL PROTEIN PRECURSOR PERFORM DISCRETE FUNCTIONS IN THE INITIATION OF POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS......................................................................... 103Introduction................................................................................................................... ........103Results...................................................................................................................................105Mutations Which Prevent the Production of Active 3Dpol are Rescued by 3CDpro.......105Complementation of 3Dpol Deficient Mutations Re quires the Intact 3CDpro Precursor....................................................................................................................107Mutations Which Disrupt 3Cpro/3CDpro Binding to the 5CL Block RNA Replication and Affect Polyprotein Processing.........................................................108Complementation of 3Cpro/3CDpro RNA Binding Mutants Requires the Intact 3CDpro Precursor........................................................................................................ 109Complementation Between Two Functionally Distinct 3CDpro Mutants......................110High Efficiency Complementation of 3C[K 12N/R13N] Requires the P3 Precursor....112Discussion.............................................................................................................................113Active 3Dpol is Admitted to the PV Replication Complex in the Form of its Polymerase-inactive Precursor 3CDpro......................................................................113RNA Binding and Protease Activities of 3CDpro are Functionally Linked................... 114Multiple 3CDpro Peptides are Present in the PV RNA Replication Complex Used to Initiate Negative-strand RNA Synthesis.................................................................... 115The 3CDpro Bound to the 5CL is Admitted to the PV Replication Complex in the Form of its Precursor P3............................................................................................ 1166 SUMMARY AND CONCLUSIONS...................................................................................126The Role of PCBP in the Initiation of Poliovirus Negative-strand Synthesis...................... 126The (MS2)2 Protein-RNA Tethering System : Virus-Host Interaction......................... 127The (MS2)2 Protein-RNA Tethering System : Host Protein Function.......................... 127The Role of Viral Protein Precursors in the Initiation of PV Negative-strand Synthesis.....128Modeling Formation of the PV RNA Replication Complex......................................... 128Close Coupling of the Viral Life -Cycle Ensures Viral Fitness..................................... 129LIST OF REFERENCES.............................................................................................................131BIOGRAPHICAL SKETCH.......................................................................................................149

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8 LIST OF FIGURES Figure page 1-1 Poliovirus genome organization an d polyprotein processing cascade. .............................. 25 1-2 Poliovirus life-cycle...................................................................................................... .....26 1-3 Genomic circularization models fo r PV translation and replication.. ................................ 27 3-1 Diagrams of the wild-typ e and m utant 5 cloverleaf.........................................................72 3-2 The C24A mutation affects negativebut no t positive-strand RNA synthesis.................. 73 3-3 Schematic of the (MS2)2 protein-RNA tethering system..................................................74 3-4 The 5CLMS2 binds (MS2)2 fusion proteins but does not bind PCBP2.............................. 75 3-5 The (MS2)2PCBP2 fusion protein restores ne gative-strand synthesis of a 5CLMS2 RNA template....................................................................................................................76 5-6 Identification of the functional domains within P CBP2 that restore negative-strand RNA synthesis of a 5CLMS2 template RNA..................................................................... 77 3-7 Levels of protein synt hesis observed in the (MS2)2 protein-RNA tethering replication reactions.............................................................................................................................78 3-8 Characterization of the K H3 domain using the (MS2)2 protein-RNA tethering system................................................................................................................................79 3-9 The (MS2)2 fusion proteins are evenly expr essed, stable, and bind to 5CLMS2 with similar affinity............................................................................................................... .....80 3-10 The ability of the combined KH1/2 domain s to restore negative-strand synthesis does not require the m ultimerization domain............................................................................. 81 3-11 PCBP1, PCBP2, and PCBP2-KL restore negativ e-strand sy nthesis to similar levels in the (MS2)2 protein-RNA tethering system........................................................................ 82 3-12 PCBP4/4A, but not PCBP3 or hnRNP K, re stores negative-strand synthesis in the (MS2)2 protein-RNA tethering system.............................................................................. 83 3-13 All PCBP family proteins, except hnRNP-K, bind to the PV 5CL.................................. 84 4-1 Translation of a PV RNA template is a prerequisite for efficient negative-strand synthesis.. ...........................................................................................................................98 4-2 Physical ribosome transit of a template RNA is not sufficient to prom ote efficient initiation of negative-strand synthesis...............................................................................99

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9 4-3 Translation of 3D or a 3D precursor is required in cis f or efficient initiation of negative-strand synthesis.................................................................................................100 4-4 Efficient initiation of negative-strand synt hesis requires transla tion of 2B or a 2B precursor in cis .. ...............................................................................................................101 4-5 Poliovirus RNA replication requires transl ation of the 2BC-P3 polyprotein precursor in cis .................................................................................................................................102 5-1 Mutations which prevent the generation of active 3Dpol block RNA replication............ 118 5-2 Viral Precursor 3CDpro complements both 3D[G327M] and 3CD[PM] in trans ............119 5-3 Complementation of 3D[G327M] or 3CD[PM] requires the intact 3CDpro precursor.... 120 5-4 Mutations which disrupt 3Cpro/3CDpro binding to the 5CL block RNA replication....... 121 5-5 Complementation of 3C[K12N/R13N] or 3C[R84S] requires the intact 3C Dpro precursor..........................................................................................................................122 5-6 Schematic of trans com plementation using two functionally distinct mutations in 3CDpro..............................................................................................................................123 5-7 Two functionally distinct 3CDpro mutants can complement each other in trans .............124 5-8 Complementation of a 3CDpro RNA binding mutant is more efficient when P3 is provided in trans ..............................................................................................................125

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10 LIST OF ABBREVIATIONS ATP Adenosine 5 triphosphate cDNA Complimentary DNA CTP Cytidine 5 triphosphate DNA Deoxyribonucleic Acid GTP Guanosine 5 triphosphate GuHCl Guanidine Hydrochloride HeLa Human cervical carcinoma cell line IF Initiation factors (Ribosomal salt wash protein preparation) IRES Internal ribosomal entry site kDa Kilodalton NTP Nucleoside 5 triphosphate NTR Non-translated region PABP Poly(A) binding protein PCBP Poly(C) binding protein PIRC Pre-initiation replication complex pol Polymerase poly(A) Polyadenosine 5 triphosphate pro Protease RNA Ribonucleic Acid S10 Supernatant from a 12,000 x g centrifugation UTP Uridine 5 triphosphate vRNA Virion RNA wt Wild type

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PROTEIN REQUIREMENTS FOR TH E INITIATION OF POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS By Allyn R. Spear August 2009 Chair: James Bert Flanegan Major: Medical Sciences Bioc hemistry and Molecular Biology During infection, poliovirus genomic R NA acts first as a messenger RNA and subsequently serves as a template for RNA replication. Because th ese processes require exclusive use of the genome, the mechanism by wh ich this transition occurs must be carefully orchestrated. The first step in RNA replicati on is initiation of negativ e-strand RNA synthesis, however, prior to this initiati on a membrane associated viral replication complex must form, requiring key viral and cellular proteins. Using a cell-free system, experiments were performed to investigate specific cellular and viral protei n requirements for replication complex formation and subsequent initiation of negative-strand RNA synthesis. A protein-RNA tethering system was devel oped to study the involvement of cellular poly(C) binding protein (PCBP) in the initiation of poliovirus negative-strand RNA synthesis. The results of these studies showed that PCBP is essential for initiation of negative-strand synthesis, and did not require direct RNA bindi ng or multimerization. The critical domain of PCBP was identified and it was shown that mu ltiple PCBP isoforms share this activity. To investigate the viral proteins required for efficient initiation of negative-strand synthesis, a series of trans replication reactions were performed. The results of these studies implicate 2BC-P3 as the critical cis-acti ng viral protein precur sor, essential for

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12 membrane-associated replication complex form ation. This precursor would be severely trans-restricted by its association with membra nes and its rapid processing, accounting for the dramatic increase in RNA replication efficiency of RNAs which generate the 2BC-P3 precursor in cis. Another viral protein precursor, 3CDpro, is also critical for many aspects of viral replication. It has multiple functions, includi ng polyprotein processing, RNA binding, and as the precursor for the polymerase (3Dpol). To investigate th e function(s) of 3CDpro involved in the initiation of negative-strand RNA synthesis, poliovirus RNAs containing distinct functional mutations within the 3CD coding region were assa yed for their ability to be complemented by either wild type or mutant 3CD proteins. The results of these studies indicate the presence of two or more molecules of 3CDpro in the replication complex, and also clearly show that active polymerase must be delivered to th is complex in the form of 3CDpro or a larger precursor.

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13 CHAPTER 1 BACKGROUND AND SIGNIFICANCE Poliovirus Pathogenesis and Epidemiology Polioviru s (PV), the causative agent of poliomyelitis, is a member of the family Picornaviridae, in the genus enterovirus. The viruses of the family Picornaviridae are small (25-30 nm diameter), non-enveloped, icosahedral (T=3), positive-sense ssRNA viruses, and can cause a variety of human diseases, including meningitis, encephalitis, poliomyelitis, pancreatitis, myocarditis, rhinitis, and hepatitis PV is spread via the fecal-or al route, typically by ingestion of contaminated food or water. The primary site of replication is in the mucosal lining of the oropharyngeal and intestinal tr act, either in epithelial or lymphoid cells, and during acute infection, virus is shed at high levels in the feces of infected individuals (171). Infectious virus is very stable in the environment, persisting in contaminated groundwater for 3-6 weeks or more (81). After primary infection in the alimentary tract, PV may enter the central nervous system by one of two routes, either by infection of a peripheral nerve and subseque nt retrograde intraaxonal transport or by crossing the bloodbrain barrier following viremia (171). Paralytic poliomyelitis occurs in 0.5-1% of infected individuals and is a direct result of the deat h of motor neurons in the spinal cord and/or motor cortex caused by infection and lysis from PV infection. Due, in part, to its extensive study following the poliomyelitis epidemics in the mid 20th century, PV has become the prototypical member of the Picornaviridae for studying the molecular mechanisms of viral replication. Although paralytic polio has probably affected mankind throughout much of recorded history, the epidemics of infantile paralysis in the early to mid 20th century are what most people associate with the concept of poliomyelitis. It was the widespread, devastating nature of this disease that spurred scientists around the world to further characterize the infectious agent and

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14 develop effective vaccines. The success of the vaccination campaign, and the lack of an animal reservoir, led the World Health Organization (W HO) to attempt global eradication set to be completed by the early 21st century. In the course of the 20 year campaign, the cost of the Global Polio Eradication Initiative (GPEI) ha s exceeded six billion dollars, and despite tremendous progress, poliomyelitis has recently b een on the rise in areas in which poliovirus remains endemic (3, 4, 177). The Americas we re the first region cer tified polio-free by the WHO in 1994, followed by the Western Pacifi c in 2000, and Europe in 2002 (69). Unfortunately, a few regions in Africa and Asia have resisted the best efforts by the GPEI, specifically Nigeria, Afghanist an, Pakistan, and India, and all cases of wild poliovirus transmission since 2006 can be traced back to virus export from one of these four countries (3). The lack of success in these regions can be attributed to multiple factors, not the least of which are inhospitable socio-political climates, adve rse geography, and an as yet unexplained variance in the host immune response to vaccination (69). In Nigeria, as a result of rumors of vaccine-induced infertility, vaccination covera ge dropped dramatically in 2002-03, leaving an even larger proportion of the population susceptibl e. Additionally, due to apparent success in northern India, aggressive vaccination campaigns were scaled back beginning in 2002. By 2006, 20 countries that had previously been polio-free reported importation of Nigerian polioviruses, 3 polio-free countries reported case s of Indian poliovirus, and wo rldwide cases had risen to over 2000 (1-3, 174). This number has not changed significantly since 2006 and worldwide incidence of poliovirus remains between 1000 and 2000 cases pe r year, with exportation of poliovirus from endemic countries remaining a serious concern. The WHO and GPEI has recently recommitted itself to the campaign, setting new deadlines fo r polio eradication: India, Afghanistan, and Pakistan by 2010, Nigeria by 2011 (177). The prosp ect of nearing eradication presents new

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15 challenges and generates some interesting scient ific questions: Since the use of oral polio vaccine (OPV) has an associated risk of cau sing vaccine derived poliomyelitis, how will the OPV be phased out and the inactivated polio va ccine (IPV) be phased in? Will the immune responses and vaccine coverage attainable using the IPV be su fficient to protect the worlds population? Will we ever be able to stop vacc ination for poliovirus? In the process of discussing these issues, the field has determined that the development of anti-polioviral drugs would be a significant benefit to public health, particularly during the transition into a polio-free world (reviewed in 59). To date, there have be en no such drugs that have shown any clinically promising results, indicative of a need for a better understanding of th e molecular biology of poliovirus replication and the identifi cation of new potential drug targets. Poliovirus Molecular Biology The PV RNA genom e (Figure 1-1A) is ~ 7.5 kilobases long, uncapped, and covalently linked to a 22 amino acid viral protein 3BVPg (v iral p rotein g enome-linked; VPg) via a phosphodiester linkage between O4 on tyrosine 3 of VPg and the 5 phosphate on the genome terminal uridine (10, 120, 179). This VPg moie ty is quickly removed by a cellular unlinking activity (11) and is not required for viral translation (166). Base s 1-89 of the 5 non-translated region (NTR) of PV genomic RNA form a cis -acting structure known as the 5 cloverleaf (5CL) which is required for genomic replication (13, 26, 93, 126, 196), RNA stability (144), and optimal translation (78, 158, 187; Ogram et al. unpublished results). More recently, a conserved cytidine-rich sequence (poly(C) tract) in the 12 nucleotides adjacent to the 5CL was identified and shown to be required for RNA replication (2 05). The remainder of the 5NTR contains a highly structured region which functions as a type-1 internal ribosomal entry site (IRES), driving cap-independent initi ation of translation (97, 164). Downst ream of the IRES is a single open reading frame encoding viral capsid and non-structural proteins as a single large polyprotein

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16 which is processed by viral proteases (Figure 1-1B) (113). The 3 NTR of PV genomic RNA contains additional cis -acting structures which, while not ab solutely essential (201), are required for efficient RNA replication (100, 138, 168). Fina lly, the genomic RNA terminates in a poly(A) tail of heterogeneous length (~90 nts) (218) which enhances IRES translation (32, 136, 192, 193) and is required for PV RNA infectivity (190). Attachment and Entry The PV lif ecycle initiates with the attachment of virions to the Ig-like cell surface receptor CD155/Pvr (Figure 1-2, Step 1)(111, 134)via su rface residues on the PV capsid in a region known as the canyon which surrounds a vertex on the icosahedral particle (87). The capsid then undergoes conformational changes whereby th e myristoylated N-terminus of VP4 and the hydrophobic N-terminus of VP1 insert into the ce ll-membrane, forming a pore structure and a membrane anchor (41, 42, 75). Recent work by Brandenburg et al showed that PV internalization into HeLa cells by endocytosis ut ilizes a clathrin-, calveolin-, and flotillinindependent pathway, and that subsequent uncoati ng and genome release o ccurs just inside the plasma membrane at the cell periphery (39). The study further showed that PV entry and uncoating required ATP, the actin cytoskeleton, and tyrosine-kinase activity. Work by Coyne et al. showed that PV entry into polarized brain va scular endothelial cells di d require calveolin and dynamin, indicating different mechanisms of en try into different cell types (63). More interestingly, Coyne et al also showed that the interacti on of PV with CD155 triggered a signaling cascade involving tyro sine phosphorylation and dramatic rearrangements in the actin cytoskeleton which were also essential for vi ral entry and uncoating. Together, these studies indicate that receptor binding by PV serves a purpose beyond attachment, in the induction of cellular signaling cascades criti cal to establishing a cellular environment conducive to infection.

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17 Viral Translation and Po lyprotein Processing Cap-independent translation of the genomic RNA is the first intracellular step in the viral life cycle (Figure 1-2, Step 2). Because viron RNA (vRNA) does not contain a 5 7mG cap structure, it cannot undergo canoni cal cap-dependent ribosomal scanning and translation as would a typical eukaryotic mRNA. Instead, pico rnaviruses utilize a complex series of RNA secondary structures in their 5NTR called an IRES to recruit and position ribosomes in a cap-independent manner (97, 164). The PV IRES is sub-classified as a type-1 IRES based on structural characteristics, and shares homology with the IRESes of other enteroand rhinoviruses (99). Like other type-1 IRESes, the PV IRES binds a set of host cell proteins to assist in RNA folding and ribosome recruitment, including th e La autoantigen, poly(C) binding protein 2 (PCBP2), SRp20, polypyrimidine tract binding pr otein (PTB), upstream-of-N-ras protein (unr ), and eIF4G (90, 131). In addition to these co-fac tors, certain RNA elemen ts within the IRES presumably act as a scaffold for the assembly of ribosomal subunits to facilitate efficient translation initiation. Eukaryotic cells have evolved a mechanis m to further optimize the cap-dependent translation of their own mRNA by inducing a 5-3 circularization via in teractions between cap binding protein (eIF4G) and poly( A) binding protein (PABP) bound to the 3 poly(A) tail (212). In turn, there is a growing body of evidence (reviewed by (131)), as well as a significant amount of unpublished data from our lab, that indicates a similar enhancement st rategy in use by PV (Ogram et al ., unpublished results). Because the PV geno me has VPg linked to its 5 end rather than a 5 7mG cap structure, it is likely that an alte rnative protein bridge mediates the 5-3 circularization of PV genomic RNA (Figure 1-3). This pres umably involves interactions between PCBP bound to the 5CL and PABP bound to the 3 poly(A) tail, although additional

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18 and/or alternative in teractions have been propos ed, including as yet unid entified cellular proteins (131). Downstream of the IRES is the singl e open reading frame which encodes the approximately 2200 amino acid viral polyprotein. Th is large protein product is processed by the viral proteases 2A (2Apro) and 3C/3CD (3Cpro/3CDpro) and the resultant polyprotein cleavage cascade is depicted in Figure 1-1B (119, 155, 156). Initial polyprotei n processing occurs co-translationally at the boundary between the structural and non-structural proteins by 2Apro (206), and all subsequent cleavag e events (except at the capsid protein VP4-VP2 junction) are mediated by the other viral protea se or its immediate precursor, 3Cpro/3CDpro (113, 155). The cleavage at the VP4-VP2 junction is catalyzed by amino acids in VP0 and is induced upon RNA packaging following final capsid assembly ( 15, 27, 155). Processing of the non-structural replication proteins occurs by two distinct path ways determined by the site of primary cleavage by 3Cpro/3CDpro (119). If the primary cleavage occurs at the 2C-3A junction, processing of the resultant P2 and P3 precursors proceeds in the soluble phase, and proceeds very slowly. However, with 3-fold higher frequency, the primary cleavage occurs at the 2A-2B junction, shunting the resultant 2BC-P3 precursor into the rapid processing membrane associated pathway. This is of particular importance given th at PV infection induces dramatic membrane rearrangements which are essentia l for PV replication (44, 45). Virus-Induced Alteration of Host Cell Environment The rem arkable rearrangement of intracellula r membranes observed during PV infection is dependent on PV translation and results in the formation of rosette-like vesicles to which PV replication complexes localize (44, 45, 70). It has been shown th at viral protein precursor 2BC and viral protein 2CATPase are responsible for these rearrangements (9, 20, 50). In addition, 2BC and 2CATPase, as well as 3AB, have been shown to affect membrane permeability and

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19 nucleo-cytoplasmic trafficking (8, 30, 31, 84, 117). The collective effect of these activities increases the cytoplasmic availability of nuclear factors utilized in PV replication and also generates the ideal membrane microenvironment which is essential for replication complex assembly and RNA replication. In addition to the alteration of host cell membranes, PV also induces a global down-regulation of host-ce ll translation and transcription. Beyond their role in PV polyprotein processing, 2Apro and 3CDpro/3Cpro are also responsible for the comprehensive proteolytic attack on critical cellular proteins and processes th at causes this host shut-off. The host cell translational machinery is primarily disabled by 2Apro cleavage of eIF4GI/II, an essential component of the cap binding complex eIF4F (72, 82, 125). In addition, both 2Apro and 3CDpro/3Cpro cleave poly(A) binding prot ein (PABP), another cellular protein involved in stimulating cap-dependent tr anslation (101, 116). Transcriptional machinery is also proteolytically inactivated by 2Apro cleavage of TATA-binding protein (TBP) (214), as well as 3CDpro/3Cpro cleavage of TBP (55), cAMP responsive element binding prot ein (CREB) (215), Oct-1 (216), and multiple other transcription fact ors specific for RNA polymerase II (181), and RNA polymerase III (54, 184). The net effect of these cleavage ev ents is a shut-off of over 95% of host cell gene expression by three hours post-infection (102). Host Protein Involvement in RNA Replication Both PCBP and PABP, discusse d above relative to PV transl ation, have also been shown to be involved in other aspect s of PV replication. As previ ously discussed, the PV genom e contains an essential RNA structure at the 5 en d, so-termed the 5 cloverleaf (5CL). PCBP binding to a stem-loop within this structure has been shown to be involved in RNA replication (12, 13, 158, 209), as well as in stabilizing PV RNA ( 144). In addition to its association with the

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20 5CL, PCBP also binds to a conserved cytidinerich sequence adjacent to the 5CL and this interaction was also shown to be required for RNA replication (205). At the opposite end of the PV genome, the 3 po ly(A) tail is of suff icient length to bind cellular PABP. It has been establ ished that the poly(A) tail plays a role in stabil ity, translation, and RNA replication, most likely as a result of its interaction with PABP (93, 182, 190; Ogram et al. unpublished results) Moreover, Silvestri et al showed that the PABP/poly(A) requirement was specific for the initiation of negative-strand RNA synthesis (186). Negative-Strand RNA Synthesis The trans ition from PV translation to genom ic RNA replication, as with all positive-strand RNA viruses, must initiate with the synthesis of anti-genomic negative-strand RNA (Figure 1-2, Step 3. As discussed previously, membrane vesi cles are essential for RNA replication since the cytoplasmic surface of these membranes is the site of replication complex assembly (34, 51). In addition to membrane vesicles, RNA replication re quires viral proteins as well as some forms of their precursors. Of all the vira l proteins and precursors describe d to date, it has been shown that 2B, 2CATPase, 3AB, VPg(3B), 3Cpro/3CDpro, and 3Dpol are essential for R NA replication (12, 175, 213). Additional data generated from complementation analysis has indicated that optimal replication may require larger precursor forms of one or more of these essential proteins (104, 124, 204). The precise functions of 2B and 2CATPase in RNA replication are unknown, however, negative-strand initiation is blocked by millimolar concentrations of guanidine HCl (GuHCl) and GuHCl resistance mutations map to 2CATPase (23, 170). The requirement for 3AB may arise from a need to provide the essential VPg protein prim er to the replication complex in a membrane associated precursor form (46). Data from our lab has suggested that the RNA dependent-RNA polymerase (3Dpol) must also be delivered to the replicat ion complex in the form of its precursor 3CDpro, prior to processing and nega tive-strand initiation (25).

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21 The polymerase initiates negative-strand synthesi s at the extreme 3 end of the viral RNA, using the poly(A) tail as a template and VPg as a protein primer. In addition to the presence of the PABP-poly(A) RNP complex, additional inter actions occur on the adjacent 3NTR, including recruitment of 3CDpro/3Dpol and 3AB/VPg to provide the activ e polymerase and protein primer, respectively (89). Interestingly, despite its loca tion at the opposite side of the genome, an RNP complex formed on the 5CL has also been shown to be essential for R NA replication (12, 13). This complex consists of both cellular PCBP as well as the viral 3CDpro (12, 158, 209). The involvement of both distal and pr oximal RNA elements in negative-strand initiation lead our lab and others to propose the formation of a 5-3 circular RNP complex, dr iven by protein-protein interactions between RNP comple xes at the 5CL and 3NTR/pol y(A) tail (Figure 1-3) (26, 93, 126, 196). Potential bridging interactions could involve a PCBP and PABP interaction, as has been shown to occur on the 3 end of -globin mRNA (49, 210). A 3CDpro-3CDpro interaction could also either drive or augm ent circularization, since the vi ral precursor has been shown to bind to both 5 and 3 ends (12, 89), and intera ction surfaces were identified in the crystal structure of 3Dpol and 3CDpol (86, 130, 153) Regardless of which is the critical interaction, the formation of the complete circular RNP complex re presents the last pre-re plication state of viral genomic RNA. Once the complex has formed and subsequent processing of any necessary protein precursors is complete, 3Dpol initiates RNA replication vi a uridylylation of VPg on the poly(A) tail. This would then be followed by elongation of negative-strand RNA, generating a full-length double-stranded re plicative form (RF) RNA. Positive-Strand RNA Synthesis Poliovirus RNA replication is highly asym m etric, generating 10-100 molecules of VPg linked positive-strand RNA for every one negativ e-strand template synt hesized (Figure 1-2,

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22 Step 4) (150, 207). The requirements for th e initiation of positive-strand synthesis differ significantly from those for negative-strand initiation. First among these differences is the requirement for pre-uridylylated VPg (VPgpUpU) as a primer for positive-strand elongation (141, 143). The synthesis of VPgpUpU occurs on an RNA hairpin template in the 2CATPase coding region termed the c is r eplication e lement or cre(2C) hairpin, and requires VPg, UTP, 3Dpol, 3CDpro, PCBP and the 5CL (126; Sharma et al. unpublished results). The uridylylation of VPg is templated by the first of three conserved adenosines in the loop of the ha irpin, and addition of the second uridyl residue is accomplished via a slide-back mechanism (161, 176). Interestingly, the cre (2C) dependent VPg uridylylation reaction is inhibited by GuHCl (126, 141) indicating the involvement of 2CATPase, even though positive-strand synthesis per se is not sensitive to GuHCl inhibition (23). The protein requirements for positive-strand initiation are also different from those observed for negative-strand synthesis. Despite GuHCl insensitivity, 2CATPase has been shown to bind specifically to the 3 end of the negative-strand, indicating a possible role in positive-strand synthesis (19). Data from the Semler lab has es tablished the specific bind ing of cellular protein hnRNP C to the 3 end of negative-strand RNA, and have shown it to be required for RNA replication (40, 178). Sequences at the 3 end of PV negative-strand RNA, which correspond to a potential hnRNP C binding site, have also been shown to be essential for positive-stand synthesis (183). Taken together, these data suggest a mechanis m of positive-strand initiation whereby hnRNP C, 3Dpol, VPgpUpU, and possibly 2CATPase form an RNP complex at the 3 end of the negative strand RNA (or RF RNA) to promote multiple sequential rounds of positive-strand RNA synthesis.

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23 Packaging and Release of Progeny Virions When sufficient PV capsid protein and genom ic RNA have been synthesized, virion assembly begins (Figure 1-2, Step 5). Ther e is no known packagi ng signal or sequence requirement for encapsidation of PV RNA, ho wever through exhaustive study of defective interfering (DI) particles and induced genomic deletions, it has been determined that the capsid coding region is not required for vRNA encapsidation (52, 115, 148). Additional studies have shown that although not essential, PV IRES se quences do enhance encapsidation (103). There also appears to be a very strict discri mination in RNA polarity, since packaging of negative-sense RNA is undetectable (150). In ad dition to sequence requirements, all packaged RNAs must be VPg linked (149), and there also appears to be a tight coupling between active RNA replication and encapsidation of nascent vRNA (152). Once the RNA is encapsidated, VP0 undergoes processing to generate VP4 and VP2, mediated by catalytic residues in VP2 and activated by the presence of RNA, which results in formation of the final infectious virus particle (15, 27, 91, 94). Release of viral particles from infected cells can occur by multiple mechanisms, including programmed cell death (203), cytopathic effect (CPE) induced lysis (7), and a utophagosome-mediated exit w itho ut l ysis (AWOL) (195). Although poliovirus infection induces pro-apoptotic programs, the programs are quickly suppressed by viral proteins (203). In fact, it is the interaction of PV with its receptor (CD155) that induces c-J un N H2-terminal k inase (JNK) activation, and ultimately this activation overcomes vira l protein mediated suppression and triggers cell death via Bax-dependen t mitochondrial dysregulation, cytochrome c release, and activation of the apoptotic caspase cascade (7, 16). Alternativel y, in the presence of inhibitors of apoptosis, Agol et al showed that the CPE caused by PV infection (e.g. membrane rearrangements, increase in nuclear permeability) we re sufficient to induce lysis of the host cell

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24 and release of viral particles (7). The release of small amount s of infectious virus has also been observed in the absence of cell lysis, and Taylor et al. has recently shown that this is a consequence of viral subversion of the cellular autophagy pathway resulting in the delivery of small pockets of virus-laden cytoplasm to the extr acellular space (195). In all cases, these newly formed viral particles, following re lease or lysis of the host cell, ca n now either spread to infect neighboring cells, or be shed into th e environment to await a new host. Cell-Free Replication System Shortly af ter the isolation and establishment of the HeLa cervical carcinoma cell line in 1951, HeLa cells were widely used to passage and study poliovirus (5, 194). A major breakthrough in PV mol ecular biology was the cDNA cloning a nd sequencing of the PV genome by Racianello and Baltimore in 1981 (172, 173). This was followed by another significant advance ten years later, when Molla, Paul a nd Wimmer successfully generated infectious poliovirus de novo using a cell-free replication system (140). Further optimization and characterization of this system by our lab and others has produced the cell-free HeLa S10 translation-replication system in use today (21, 22, 24). This system permits us to uncouple the otherwise intertwined processes of translation and replication, a llowing us to finely dissect the molecular mechanisms of these events while still accurate ly recapitulating in vivo viral replication. Recent developments involving the use of ribozyme generated authentic 5 ends on transcript RNAs have allowed us to further di ssect RNA replication and examine the molecular biology and genetics of negative-strand and positive-strand synthesi s separately (92, 141). Using this system, we have begun to identify the viral a nd cellular proteins requir ed for the initiation of poliovirus negative-strand RNA synthesis.

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25 Figure 1-1. Poliovirus genome or ganization and polyprotein proces sing cascade. A) PV genomic RNA is covalently linked to VPg at its 5 end, and contains multiple cis acting RNA elements, including the 5 cloverleaf (5CL) poly(C) tract, IRES, cre(2C) stem-loop, and 3NTR/poly(A) tail. B) PV translates a single large polyprotein which is processed by viral proteases 2Apro ( ) and 3Cpro/3CDpro ( ). Primary cleavage of the P23 precursor at the 2A-2B junction occurs with 3-fold higher efficiency than cleavage at the P2-P3 junction. This initial processing event determines if the subsequent processing occurs in membra ne-associated or soluble compartments.

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26 Figure 1-2. Poliovirus life-cycle. PV binds its cellular receptor CD155, undergoes internalization by endocytosis, and releases its genome into the cytoplasm at the cell periphery (Step 1). Upon release, translation factors and ribosomes assemble on the IRES and viral protein synthesis occurs (Step 2). Using these newly synthesized proteins, the viral replication complex is formed and the 3D polymerase generates a new VPg-linked negative-strand RNA (Step 3) This dsRNA intermediate is then used as a template for multiple rounds of VPg-UU primed positive-strand synthesis (Step 4). When sufficient RNA and pr otein synthesis has occurred, nascent positive-strand genomic RNAs are packaged by the viral capsid proteins and these progeny virions are released upon apoptosis of the host cell (Step 5).

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27 Figure 1-3. Genomic circularization models for PV translation a nd replication. Prior to viral protein synthesis, interactions between PCBP and PABP circularize PV genomic RNA to facilitate ribosome reloading and enhance translation. Following viral protein synthesis, replica tion protein precursors 3CDpro and 3AB would be recruited to the 5CL and 3NTR, undergo processing, and initiate VPg-primed negative-strand RNA synthesis.

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28 CHAPTER 2 MATERIALS AND METHODS DNA Manipulation and Cloning Techniques All restriction enzym es, as we ll as the Klenow fragment of T4 DNA polymerase, used in these studies were obtained from New England Biolabs unless otherwise noted. Restriction digests were performed accordi ng to manufacturers protocols, and when double digests were required they were performed sequentially unl ess optimal conditions were available for simultaneous digests. Standard PCR reactions were carried out according to the manufacturers suggested protocols using either TrueFidelity DNA polymerase (Continental Lab Products), PfuUltra Fusion II DNA polymerase (Stratagene), A ccuprime Pfx DNA polymerase (Invitrogen), or Phusion DNA polymerase (New England Biolabs). Rapid purification of PCR products for direct restriction dige st was performed using QiaQuick PCR Cleanup kit from Qiagen. Gel purification of PCR fragments or restriction enzyme digested DNA was performed using the GeneClean II Spin Kit from Bio101. For gel purification, DNA fragments were resolved on SeaKem GTG agarose (formulated for gel purifi cation) from Cambrex, visualized by ethidium bromide staining on a low intens ity UV transilluminator, and a ppropriate bands were excised using a scalpel. All vector DNAs, where re quired, were dephosphorylated using Shrimp Alkaline Phosphatase (SAP) from Ro che Applied Science. It is critical that dephosphorylation be performed after gel purification, as this will redu ce vector background to near zero. Dephosphorylation reactions were performed by adding SAP to 10% of the total reaction volume and using the provided 10X dephosphorylati on buffer [500 mM TrisHCl (pH=8.5), 50 mM MgCl2,]. Following a 1 h incubation at 37C, the SAP was inactivated by incubation at 65C for 15 min. Vector and insert DNA fragments were quantitated by agarose ge l electrophoresis and ethidium bromide visualization of each frag ment versus the 1 kb or 100 bp DNA ladder (New

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29 England Biolabs). Ligations were performed us ing T4 DNA ligase obtained from Promega. All ligations utilized the provid ed 10X ligase buffer [300 mM TrisHCl (pH=7.8), 100 mM MgCl2, 100 mM DTT, 10 mM ATP] and contained 1 Unit/ L T4 DNA ligase. All reactions contained a total of 5 ng/L DNA (vector + insert), however, for sticky-sticky and st icky-blunt ligations, a ratio of 1:3 (vector:insert) was used, whereas for blunt-blunt liga tions, a ratio of 1:1 (vector:insert) was used. Fo r sticky-sticky ligations, reacti ons were incubated at room temperature for 1 h or more prio r to transformation, and for stickyblunt or blunt-b lunt ligations, reactions were incubated at room temperature for at least 12 h or more prior to transformation. Site-Directed Mutagenesis Site-directed m utagenesis was performed using a procedure based on that of the Stratagenes QuikChange site-directed mutagene sis kit. Briefly, two complementary mutagenic primers are designed which contai n the desired mutations flanked on either side by 10-15 nts of non-mutagenic complementary sequence. Using th ese primers, the appr opriate template, and PfuTurbo DNA polymerase (Stratagene), PCR r eactions were performed where the elongation times were extended to allow complete transit around the circular plasmid DNA template. After 18 PCR cycles, 20 Units of Dpn I was added to the reaction and incubated for 1 h at 37C to digest all methylated input DNA template. Fo llowing digestion, 1 L of this reaction was transformed into XL-1 Blue competent cells (Stratagene). Resultant ampicillin resistant colonies were screened by DNA mini-prep and restriction dige st and sequencing. Once the sequence of the mutated region was verified, a restriction fr agment within the seque nced region, containing the mutation, was transferred back into the pare nt vector background to prevent accumulation of secondary vector mutations that could potential ly arise during PCR of the entire plasmid.

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30 Two-Step PCR Two-step PCR was perform ed to induce mutations (two-step mutagenic PCR) as well as to fuse two DNA sequences (two-step semi-ove rlapping PCR). For mutagenic PCR, two complementary mutagenic primers were designed which contain the desired mutations flanked on either side by 15-20 nts of non-mutagenic complementary sequence. Additionally, two outside primers were designed, one 5 of the desi red mutation and one 3 of the desired mutation. For convenience, these primers were each 3001000 bases away from the position of the mutatgenic primers. It was also essential th at these outside primers also encompassed unique restriction enzyme sites for reintroduction of th e mutated fragment. Two first step PCRs were performed using standard PCR conditions and enzymes described above. These PCRs both utilized the same template DNA, however one co ntained the primer pair to generate the 5 product and the other reaction contained the primer pair to generate the 3 product. Here, both products share the entire mutagenic primer sequence (i.e. the 3 end of one product is fully complementary to the 5 end of the other). Thes e first step PCR products were then gel purified and quantitated using methodology described above. The second step PCRs contained 10-20 ng of each first step PCR product and only the 5 an d 3 outside primers. Here, the first step products are added in place of plasmid template DNA and the mu tations are already present in these templates, so mutagenic primers are no long er necessary. All second step PCRs used only PfuUltra Fusion DNA polymerase (Stratagene) or Phusion DNA polym erase (New England Biolabs) as these were empirically determined to generate the highest product yield with the lowest extraneous background amplification. The second step PCR product was a result of priming of one first step PCR product on the othe r, followed by amplification of this combined product by the outside primer pair. The sec ond step PCR product was purified using the QiaQuick PCR purification kit a nd the resultant DNA was digested using the restriction enzymes

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31 whose sites flanked the induced mutation. The restriction fragment of the second step PCR product, which contained the desi red mutation, was then cloned in to the corresponding sites of the parent plasmid DNA. In cases where two DNA sequences needed to be fused, two-step semi-overlapping PCR was used. To do this, a sequence map of the final desired (fused) sequence was generated. Two complementary primers were then designed such that the primer pair equally spanned the junction between the two fused sequences. This re sulted in the generatio n of two primers which each contained equal halves of two distinct sequences. As with two-step mutagenic PCR described above, two additional outside primers were also required, however these primers were designed to be on the 5 and 3 sides of the fusion junction of th e desired sequence. The parent plasmid DNA for the 5 sequence to be fused was c hosen to be the recipient vector DNA, due to the availability of convenient un ique restriction sites. Therefore, the 3 outside primer was designed to include a restriction site corresponding to a site available in the vector DNA. First and second step PCRs were performed exactly as described above for two-step mutagenic PCR, except the semi-overlapping primers (primers ove rlapping the fusion junction) were used in place of the mutagenic primers. Here, the seco nd step PCR product represents a new synthetic gene fusion of the two previously distinct DNA sequences. Purified second step PCR product was digested with the appropriate restriction enzymes and cloned into the corres ponding sites of the recipient vector DNA. Construct Verification and DNA Stock Preparation Sm all scale plasmid DNA of potential clones was prepared using either the Eppendorf or Qiagen Mini-Prep Spin kits. The correctness of all constructs was verified by sequencing performed either at the DNA Seque ncing Core Laborator y (ICBR, University of Florida) or by the SeqWright commercial sequencing facility (SeqWright, Inc., Houston, TX). All primary

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32 clones generated using site direct ed mutagenesis were subsequently recloned back into the parent vector background by excision and tran sfer of a sequence verified re striction fragment containing the desired mutation. Although XL-1 Blue competen t cells (Stratagene) we re utilized for some sub-cloning applications, all fi nal plasmid DNAs were transfor med into MAX Efficiency StBL2 competent cells (Invitrogen) for preparation of gly cerol stocks as well as for large scale plasmid DNA preparation. For long term storage, 50% gly cerol stocks of overnight liquid cultures were maintained at -80C and were re-struck on LB+ampic illin agar plates as needed. For large scale plasmid DNA preparations, a single colony from a newly struck LB+ampicillin plate was inoculated into 250 mL of LB broth containing 50 mg/mL ampic illin. These inoculated broth cultures were grown overnight at 37C with sh aking, and bacterial pellets were isolated by centrifugation at 5,000 x g for 10 min. Plasmid DNA was subsequently isolated using the Qiagen Midi-Prep kit. All plasmid DNA stocks were standardized to 0.5 g/L and stored in TE [10 mM TrisHCl (pH=8), 1 mM EDTA] at -20C. cDNA Clones Used in These Studies Poliovirus Clones Used in These Studies A previously described cDNA clone of the Mahone y strain of type I poliovirus, designated pT7-PV1(A)80, was used as the parent clone for all poli ovirus based constructs used in all studies herein (26). (i) pPV1 GUA3 is a previously described construct which generates an RNA transcript [PV1 GUA3 RNA] with a 5-nt deletion in the 3 NTR, known to inhibit negative-strand synthesis without a ffecting translation ( 26). (ii) pP23 is a previously described construct with a deletion of the P1 capsid coding re gion (104). RNA transcripts of this construct [P23 RNA] express a ll essential replication proteins from the P2 and P3 regions of the viral genome. These transcripts function as an R NA replicon, allowing for negative-strand, but not positive-strand synthesis. (iii) pRzP23 was generated from pP23 by inserting a hammerhead

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33 ribozyme (Rz) downstream of the T7 promoter. Following transcription, this ribozyme removes itself from the 5 end of the transcript, yielding RNA transcripts with authentic poliovirus 5 ends (25, 141). These authentic ended transcripts func tion as replicons, capable of both positiveand negative-strand synthesis. (iv) pP23-5CL( C24A) and pRzP23-5CL(C 24A) were engineered using site-directed mutagenesis. Transcripts of these constructs contain the mutant 5CLC24A, but translate all viral replication pr oteins. (vi) pP23-5CL(MS2) wa s generated using site-directed mutagenesis, replacing nts 12-32 in pP23 (ste m-loop b of 5CL) with the cDNA for a 19 nt stem-loop from the MS2 bacteriophage genome sequence [ACATGAGGATTACCCATGT] (114). Proper folding of the resulting mutant 5CLMS2 was verified using the Mfold RNA structure prediction program (220, 221). (vii) pF3 is a previously described construct in which the P1 and P2 coding regions were deleted, and a frameshift mutation wa s engineered near the beginning of the P3 coding region (183). Transc ripts of this constr uct [F3 RNA] initiate translation at the 3A start codon but prematurel y terminate translation. (viii) pF3-5CL(C24A) was generated from pF3 using site-directed mutage nesis and transcripts of this construct contain the mutant 5CLC24A. (ix) pFS23 is derived from pP23 via the deletion of nucleotides 775-779 by restriction digest, blunting with the Klenow fragment of T4 DNA polymerase, and re-ligation. This deletion generated a reading frame shift causing transcri pts of this construct [FS23] to initiate translation at the 2A start codon but prematurely termin ate after the synthesis of a 65 amino acid peptide. (x) pP1-3D* is derived from pT7-PV1(A)80 via insertion of a Stu I restriction site at position 3364 by site-d irected mutagenesis and s ubsequent removal of the Stu I-Dra III fragment by restriction digest, blunting by T4 DNA polymerase, and re-ligation. The net effect of this process is an in-frame deletion of nuc leotides 3365-6082, spanning the extreme 3 end of the P1 coding region through a 5 portion of the 3D coding region. Transcripts of this construct

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34 [P1-3D* RNA] express a non-functional protein consis ting of the majority of the P1 precursor (amino acids 1-874) fused to a large carboxy-terminal portion of 3D (amino acids 13-460). (xi) pFS1-3D* is derived from pP1-3D* via the deletion of nucleot ides 1119-1122 by restriction digest, blunting with the Kle now fragment of T4 DNA polymer ase, and re-ligation. This deletion generated a reading frame shift causi ng transcripts of this construct [FS1-3D* RNA] to initiate translation at the VP4 start codon but prematurely terminate after the synthesis of a 133 amino acid peptide. (xii) pP23-2ASTOP is derived from pP23 via insertion of two stop codons following the terminal Gln codon of the 2A c oding sequence by site-directed mutagenesis. Transcripts of this construct [P23-2ASTOP RNA] retain the translation initiation context and all other RNA sequences of P23 RNA but translation termin ates at the precis e carboxy-terminus of 2A. (xii) pP23-2BSTOP is derived from pP23 via insertion of two stop codons following the terminal Gln codon of the 2B coding sequence by site-directed mutagenesis. Transcripts of this construct [P23-2BSTOP RNA] retain the translation initiation context and all other RNA sequences of P23 RNA but translation terminat es at the precise car boxy-terminus of 2B. (xiv) pP23-2CSTOP is derived from pP23 via insertion of two stop codons following the terminal Gln codon of the 2C coding sequence by site-directed mutagenesis. Transcripts of this construct [P23-2CSTOP RNA] retain the translation initiation context and all other RNA sequences of P23 RNA but translation terminates at the prec ise carboxy-terminus of 2C. (xv) pP23-3ASTOP is derived from pP23 via insertion of two stop codons following the terminal Gln codon of the 3A coding sequence by site-directed mutagenesis. Transcripts of this construct [P23-3ASTOP RNA] retain the translation initiation context and all other RNA sequences of P23 RNA but translation terminates at the precise carboxy-te rminus of 3A. (xvi) pP23-3BSTOP is derived from pP23 via insertion of two stop codons following the termin al Gln codon of the 3B coding sequence by

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35 site-directed mutagenesis. Tran scripts of this construct [P23-3BSTOP RNA] retain the translation initiation context and all other RNA sequences of P23 RNA but translation terminates at the precise carboxy-terminus of 3B. (xvii) pP23-3CSTOP is derived from pP23 via insertion of two stop codons following the terminal Gln codon of the 3C coding sequence by site-directed mutagenesis. Transcripts of this construct [P23-3CSTOP RNA] retain the translation initiation context and all other RNA sequences of P23 RN A but translation terminates at the precise carboxy-terminus of 3C. (xviii) pP V1p50 is derived from pT7-PV1(A)80 via an in-frame deletion of nucleotides 867-6011 by digestion with BstB I and re-ligation of the vector fragment. Transcripts of this construct [PV1p50 RNA] u tilize the authentic transl ational start and stop contexts and express a non-functional fusion protein (p50) between a short VP4 peptide (amino acids 1-41) and the majority of 3D (amino acids 9-460). (xix) p2BC-P3 is a previously described construct which contains the coding region for th e 2BC-P3 precursor protein (104). Transcripts of this construct [2BC-P3 RNA] express the 2BC-P3 precursor protein. (xx) p2C-P3 is derived from two previously described constr ucts p2C and pP23 via removal of the BamHI fragment from p2C and subsequent ligati on of that fragment into the corresponding sites of pP23 (104). Transcripts of this construct [2C-P3 RNA] expr ess a 2C-P3 precursor protein. (xxi) pP3 is a previously described construct which contains the coding region for the entire P3 polyprotein precursor (104). Transcripts of this construct [P3 RNA] express the P3 polyprotein precursor. (xxii) p3BCD is derived from pP3 via site dir ected mutagenesis and subsequent removal of the 3A coding region. Briefly, a Sma I restriction site, Kozaks co nsensus sequence and AUG codon were introduced into pP3 immediately 5 of th e initiating Gly codon of 3B. This mutant pP3 now contained tandem Sma I sites, one immediately upstream of the P3 start codon, and one immediately prior to the newly introduced 3B start codon. This subclone was digested with

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36 Sma I, removing the 3A coding region, and subsequen tly re-ligated to form p3BCD. Transcripts of this construct [3BCD RNA] express the 3BCD precursor protein. ( xxiii) p3CD is derived by an in-frame insertion of the 3C-3D(nt 1-247) protein coding sequence into the Msc I site of a previously described vector, pDJB2 (104). This v ector retains the 5 and 3NTR/poly(A) tail of full-length cDNA clone pT7PV1(A)80, as well as a portion of 3D coding region, and contains the Kozaks consensus translational start site. Tran scripts of this constr uct [3CD RNA] express proteolytically active 3CDpro precursor protein. (xxiv) p3D is derived by an in-frame insertion of the 3D(nt 1-247) protein coding sequence into the MscI site of pDJB2, as described above. Transcripts of this construct [3D RNA] express active 3Dpol alone. (xxv) To ge nerate a series of PV polyprotein expression RNAs that w ould act as partial helper RNAs, the AvrIIMlu I fragment from pPV1 GUA3 was transferred into the corresponding sites of p3D, p3CD, pP3, and p2BC-P3, generating p3D GUA3, p3CD GUA3, pP3 GUA3, and p2BC-P3 GUA3, respectively. Each of these ge nerates transcripts which express the indicated portion of the PV polyprotein but are defective for RNA replication. (xxvi) p3CD[3D-G327M] GUA3 and pP23[3D-G327M] were created by transferring the BstB I-Avr II fragment (containing the G327M mutation) to p3CD GUA3 or pP23, from pT7-PV1(A)80[3D-G327M] which had been generated previously in our laboratory. Transc ripts of these cons tructs [3CD(G327M) GUA3 RNA or P23[3D-G327M] RNA] express 3CD which pro cesses and binds RNA normally, however, the G327M mutation has disrupted the essential YGDD catalytic RNA polymerase motif, abolishing polymerase activity (98) (xxvii) p3CD[PM] GUA3 was created by mutagenic two-step PCR, using p3CD GUA3 as a template. This mutant combin es two previously described processing site mutations [T181K, Q182D] with two additional mutations [S183G, Q184N] designed to completely abrogate 3C-3D processing (12, 37, 88). Transcripts of this construct

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37 [3CD(PM) GUA3 RNA] express 3CD which retains R NA binding and protease activity, but is unable to be processed into 3C and active 3Dpol. (xxviii) pP23[3CD(PM)] was created by transferring the Bgl IIBstB I restriction fragme nt from p3CD[PM] GUA3 into the corresponding sites of pP23. (xxix) p3CD[3C-R84S] GUA3 was created by mutagenic two-step PCR, using p3CD GUA3 as a template. This mutation was previ ously identified to inhibit the RNA binding ability of 3Cpro (36). Transcripts of this construct [3CD(R84S) GUA3 RNA] express a 3CD with impaired RNA binding abi lities, but retains the ability to release a fully wild-type 3Dpol. (xxx) pP23[3C-R84S] was created by transferring the Bgl IIBstB I restriction fragment from p3CD[PM] GUA3 into the corresponding sites of pP23. (xxxi) p3CD[3C-K12N/R13N] GUA3 and pP23[3C-K12N/R13N] were created by mutagenic two-step PCR, using p3CD GUA3 or p23 as templates, respectively. These mutations were previously identif ied to inhibit the RNA binding ability of 3Cpro (36). Poliovirus-Based Protein Expression Clones Used in These Studies As described above, pDJB2 containing the GUA3 mutation was used as a PV expression vector to direct translation of a downstream reading frame (104). As before, all protein expression clones generate GUA3 RNA transcripts, which pr events the transcripts from functioning as RNA replicons (26). Th is vector can be digested with Msc I which cuts directly downstream from the IRES, prior to any initiating AUG codons. PCR products containing the Kozaks consensus translation initiation sequence and the coding region of a gene of interest can then be ligated into this vector, effectively replacing the PV coding sequence with that of the desired protein. (i) cDNA clones containing the PCBP1 and PCBP2 coding sequence were generously provided by Dr. Raul Andino (77). The PCBP1 and PCBP2 coding sequences were PCR amplified from plasmid DNA and cloned into the MscI site of pDJB2 GUA3 as described above, generating the PCBP1 and PC BP2 expression constructs pPCBP1 GUA3 and

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38 pPCBP2 GUA3. (ii) The MS2 coding sequence (137) was synthesized by GeneArt, optimizing codon usage for both mammalian and bacterial e xpression. The synthetic DNA contained a 5 Sma I site as well as consensus Kozaks sequence upstream of the initiating AUG. For expression and cloning purposes, the MS2 coding sequence was tailed with a 3 RGSH linker (112) and the 5 nucleotides of the P CBP2 coding sequence through the preexisting Ale I site. This synthetic MS2 DNA was dige sted and cloned into the Sma I and Ale I sites of the pPCBP2 GUA3 expression construct, generating an in-frame fusion of MS2 and PCBP2 (pMS2-PCBP2 GUA3). Using two-step PCR, a second MS2 coding region was fused, in-frame, upstream of the original MS2-PCBP 2 sequence, generating the p(MS2)2PCBP2 GUA3 expression construct, in which the two MS2 seque nces were joined with a GAPGIHPGM peptide linker, described by Hook et al (96). (iii) The (MS2)2 expression construct [p(MS2)2 GUA3] was generated by introducing two stop codons in the (MS2)2PCBP2 GUA3 plasmid, following the RGSH linker sequence, using site-directed mutagenesis. The PCBP2 coding sequence was then removed from this construct by restrict ion digest and re-ligation. (iv) The (MS2)2PCBP1 expression construct [p(MS2)2PCBP1 GUA3] was generated by semioverlapping two-step PCR using p(MS2)2 GUA3 and pPCBP1 GUA3 as templates. The resultant fused PCR product was ligated into the Xma I and XhoI sites of p(MS2)2 GUA3. (v) The PCBP2-KL and (MS2)2PCBP2-KL expression constructs [pPCBP2-KL GUA3 and p(MS2)2PCBP2-KL GUA3] were generated by deleting the cDNA corresponding to exon 8a in each parent clone using two-step mutagenic PCR. (vi) The (MS2)2KH1[Region] expression construct [p(MS2)2KH1[Region] GUA3] was generated by PCR amplifica tion of the coding sequence for amino acids 1-91 of PCBP2 using a 5 phosphorylated (5PO4) primer and a 3 primer containing an XhoI site. This product was digested with Xho I and ligated into the Ale I and XhoI sites of

PAGE 39

39 p(MS2)2PCBP2 GUA3, essentially replacing the PCBP2 coding sequence downstream of the RGSH linker. (vii) The (MS2)2KH2[Region] expression construct [p(MS2)2KH2[Region] GUA3] was generated by PCR amplifica tion of the coding sequence for amino acids 90-233 of PCBP2 and subsequent cloning into the XhoI and AleI sites of p(MS2)2PCBP2 GUA3, as described above. (viii) The (MS2)2KH2[Region] expression construct [p(MS2)2KH3[Region] GUA3] was generated by PCR amplification of the coding sequence for amino acids 234-364 of PCBP2 and subsequent cloning into the XhoI and AleI sites of p(MS2)2PCBP2 GUA3, as described above. (ix) The (MS2)2KH1/2[Region] expression construct [p(MS2)2KH1/2[Region] GUA3] was generated by PCR amplification of the coding sequence for amino acids 1-233of PCBP2 and subsequent cloning into the XhoI and Ale I sites of p(MS2)2PCBP2 GUA3, as described above. (x) The (MS2)2KH3[Domain] expression construct [p(MS2)2KH3[Domain] GUA3] was generated by PCR amplifica tion of the coding sequence for amino acids 269-357 of PCBP2 and subsequent cloning into the XhoI and AleI sites of p(MS2)2PCBP2 GUA3, as described above. (xi) The (MS2)2KH31 expression construct [p(MS2)2KH31 GUA3] was generated by PCR amplification of the coding sequence for amino acids 280-357 of PCBP2 and subsequent cloning into the XhoI and AleI sites of p(MS2)2PCBP2 GUA3, as described above. Transcripts of this construct express an (MS2)2 fusion to the KH3 domain with the first -strand of the domain deleted. (xii) The (MS2)2KH33 expression construct [p(MS2)2KH33 GUA3] was generated by PCR amplification of the coding sequence for amino acids 269-340 of PCBP2 a nd subsequent cloning into the XhoI and Ale I sites of p(MS2)2PCBP2 GUA3, as described above. Transcripts of this construct express an (MS2)2 fusion to the KH3 domain with the last -helix of the domain deleted. (xiii) The (MS2)2KH1/2MD expression construct [p(MS2)2KH1/2MD] was

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40 generated by deleting the coding sequence for amino acids 125-158 of PCBP2 from the context of p(MS2)2KH1/2[Region] using two-step mutagenic PCR. Transcripts of this construct express an (MS2)2 fusion to the amino-terminal KH1/2 frag ment of PCBP2 with a deletion in the multimerization domain of KH2. To obtain cDNAs for the remaining PCBP family members (PCBP3, PCBP4, PCBP4A, and hnRNP-K), total cellular mRNA was obtained from 106 HeLa S3 suspension cells using TRIzol Reagent (Invitrogen) and subsequent etha nol precipitation. Usi ng this purified cellular mRNA, 1st-strand cDNA was generated using SuperScrip t III Reverse Transcriptase (Invitrogen) with either Oligo(dT)20 or DNA primers complimentary to the 3NTR of the gene of interest. Digestion of the parent mRNA was ach ieved using RNase H (Invitrogen) and 2nd-strand cDNA was generated using PfuUltra Fusion Polymerase (S tratagene) with primers complimentary to the 5NTR of the gene of interest. This newl y synthesized cDNA was further amplified using PfuUltra Fusion Polymerase (Stratagene) and 5PO4 versions of the same 5 and 3 primers as were used for 1st and 2nd-strand cDNA synthesis. These specific PCR products were subcloned into the EcoR V site of pLITMUS39 (New England Biolab s) for blue/white screening. Following restriction enzyme screening, each coding sequence was PCR amplified from a correct subclone using 5 and 3 PO4 primers specific to the precise gene st art and stop. In addition, the 5 primer contained the Kozaks consensus sequence immediately prior to the initiating ATG, and the 3 primer contained an additional stop codon to prev ent any potential translational read-through. In all cases, the PCR products were ligated into the MscI site of pDJB2 GUA3, generating the expression constructs pPCBP3 GUA3, pPCBP4 GUA3, pPCBP4A GUA3, and pHnRNP-K GUA3. The coding sequence for each of th ese PCBP family members was further fused to the (MS2)2 coding sequence by semi-overlapping tw o-step PCR and ligation into the

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41 Xma I and XhoI sites of p(MS2)2 GUA3. This resulted in the generation of the expression constructs p(MS2)2PCBP3 GUA3, p(MS2)2PCBP4 GUA3, p(MS2)2PCBP4A GUA3, and p(MS2)2hnRNP-K GUA3. Bacterial Protein Expression Clones Used in These Studies To inducibly express proteins of intere st, the pET16b plasm id DNA was obtained from Novagen. Although this vector contains sequences encodi ng a previously inserted amino-terminal polyhistidine tag, these sequences were removed as a result of the cloning process. This would then result in the expres sion of fully wild-type, untagged protein following transfection and induction in expr ession cells. (i) To generate th e bacterial expression constructs for PCBP1, PCBP2, PCBP2-KL, and PCBP3, the c oding sequence for each protein was removed from the corresponding poliovirus based expr ession clone (described above) using NcoI and XhoI The resultant fragment was ligated into the corresponding sites of similarly digested pET16b plasmid DNA, generating pET16-P CBP1, pET-PCBP2, pET16-PCBP2KL, and pET16-PCBP3. (ii) To generate the bacteria l expression constructs for PCBP4 and PCBP4A, each coding sequence was amplified from each of the poliovirus expression constructs using a 5 primer containing a BspH I restriction site and a 3 primer containing an XhoI restriction site, since BspH I generates an NcoI-compatible overhang. Each PCR product was digested with BspH I and XhoI, and subsequently ligated into an NcoI/XhoI digested pET16b vector DNA, generating pET16-PCBP4 and pET16-PCBP4A. (i ii) To generate the bacterial expression construct for hnRNP-K, pET16b was digested with XhoI, filled in with the Klenow fragment of T4 DNA polymerase, and then further digested with NcoI. The entire coding sequence for hnRNP-K was removed from the poliovirus expression construct by digestion with NcoI and Pme I (blunt cut). This fragment was subsequen tly ligated into the corresponding sites of the NcoI/XhoI-blunt pET16b vector DNA, generating pET16-hnRNPK

PAGE 42

42 RNA Transcript Preparat ion and Purification Prior to in v itro transcription, the run-off transcription template was prepared by digesting the desired plasmid DNA with Mlu I. Digestion with this enzyme resulted in linearization of the circular plasmid DNA via a single cut immediately following the poliovirus 3NTR/poly(A) tail. Restriction digest reactions were phenol:chloroform extracted three times, chloroform extracted three times, and subsequently ethanol precipitated. Ethanol precipitated Mlu I cut template DNA was resuspended in TE, standardized to 0.5 g/L, and stored at -20C. Standard Transcription Standard transcription conditions w ere us ed for generating a ll non-capped, non-ribozyme transcript RNAs. In these conditions, transcript ion reactions contained 1X transcription buffer [40 mM TrisHCl (pH=8), 6 mM MgCl2, 2 mM spermidine], 10 mM DTT, 0.4 U/L RNasin (Promega), 1000 M of each NTP (ATP, CTP, GT P, UTP), and 15 ng/L linearized template. Bacterially expressed recombinant T7 polym erase was purified by B. Joan Morasco, and approximately 1 L of this purified T7 polymer ase was used per 100 L transcription reaction. Reactions were incubated at 37C for 2 h and were stopped by the addition of 2.5 volumes of 0.5% SDS buffer [10 mM TrisHCl (pH=7.5), 100 mM NaCl, 1 mM EDTA, 0.5% sodium dodecyl sulfate]. For purification purposes, RNA transcripts were phenol:chloroform extracted three times, chloroform extracted three times, and subsequen tly precipitated by the ad dition of three volumes of 100% ethanol and incubation overnight at -20C. Precipitated RNAs were further purified by desalting over Sephadex G-50 (GE Healthcare) gel filtra tion resin (0.5 x 15 cm column). Peak fractions containing RNA were id entified and quantitate d spectrophotometrically. All fractions containing significant quantities of RNA were pool ed, aliquotted (10-20 g /aliquot), and ethanol

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43 precipitated. These purified, desa lted transcript RNAs were store d, in ethanol, at -20C and were only precipitated immediately prior to their us e in translation/replication experiments. Ribozyme Optimized Transcription For RNA transcripts containing the Rz sequence, transcription conditi ons were altered to optim ize Rz cleavage (141). Thes e conditions are identical to thos e provided above for standard transcription, with the exception of the NTP c oncentrations. Here, each NTP (ATP, CTP, GTP, UTP) was included in the transc ription reaction at 500 M. It was determined experimentally that these conditions resulted in >95% ribozym e cleavage efficiency. Ribozyme optimized transcription reactions were st opped, extracted, desalted, and st ored in the same manner as described above for standard transcription reactions. 5 Capping Transcription To synthesize RNAs with a m7G cap analog, transcription conditions were slightly altered to optimize the capping reaction. These conditions are identical to those provided above for standard transcription, with th e exception that the GTP concentration was lowered to 200 M, and 800 M of m7G[5]ppp[5]G cap analog (Epicentre) was added to the tran scription reaction mixture (26). Under these conditions, ~80% of the transcript RNAs contain a 5 7mG cap. Capping transcription reactions we re stopped, extracted, desalted, a nd stored in the same manner as described above for standa rd transcription reactions. HeLa Extract Preparation HeLa S3 cells were ad apted to liquid suspension culture and were maintained in Jokliks modified Eagle medium supplemented with 5% bovine calf serum (Hyclone) and 2% FetalClone II (Hyclone). Cells were passaged as needed to mainta in a cell density of less than 5 x 105 cells/mL. In cases where ce lls were being grown for the purposes of an S10 preparation, HeLa cells were pelleted and resuspended in fresh 18-24 h prior to their use in the S10

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44 proceedure. The following procedures were originally developed in our laboratory by Barton et al and are described in more deta il in previous publications (21, 22, 24). Any deviation from these published procedures has been noted, where applicable. S10 Preparation HeLa cell density was determ ined by he mocytometer count, and approximately 109 HeLa suspension cells were pelleted by low speed centrifugation. This ce ll pellet was washed sequentially with 2 L of isotonic buffer [ 35 mM HEPES-KOH (pH=7.4), 146 mM NaCl, 5 mM dextrose], and was resuspended over 10 min on ice in 1.5 volumes of hypotonic buffer [20 mM HEPES-KOH (pH=7.4), 10 mM KCl, 1.5 mM Mg(CH3CO2)2, 1 mM DTT] with gentle vortexing. Resuspended swollen cells were tran sferred to a glass dounce homogenizer and were dounced on ice using a type A or tight pestle. Cell integrity was monitored by removing a small aliquot at various times during douncing and visually assessing percent lysis by light microscopy. Optimal lysis was defined as approxim ately 80% lysis with visibly intact nuclei and typically required 20-25 strokes of the dounce. The final volume of this mixture was determined and 1/9 volume of 10X S10 buffer [200 mM HEPES-KOH (pH=7.4), 1.2 M K(CH3CO2), 40 mM Mg(CH3CO2)2, 50 mM DTT] was added to make the fina l solution 1X. Following this addition, unlysed cells, nuclei, and othe r dense debris were removed by low speed centrifugation. The resultant semi-cleared supernatant was subsequen tly transferred to a sili conized corex tube and centrifuged at 12,000 x g for 15 min at 4C. The s upernatant from this ce ntrifugation was treated with micrococcal nuclease (5 g/mL, 20C, 15 min) in the presence of CaCl2 (1 mM) to degrade all endogenous cytoplasmic cellu lar mRNAs. After micrococ cal nuclease treatment, the nuclease was inactivated by additi on of EGTA (2 mM) to chelate the essential calcium. Any additional insoluble debris wa s the removed by a second 15 min centrifugation at 12,000 x g. The supernatant from this spin was divided into single use aliquots and stor ed at -80C. This

PAGE 45

45 supernatant is defined as HeLa S10 for the purpo ses of all experiments performed in the studies herein. IF Preparation The initial p rocedure for IF preparation is exactly the same as described above for S10 preparation with three significant changes: 1)Washed cell pellets were resuspended in 2 volumes of hypotonic buffer, rather than 1.5 volumes. 2)Swollen cells were dounced to 90-100% lysis, rather than 80%, however minimal disruption of nuclei is still ideal. 3) The supernatant from the first 12,000 x g centrifugation is not tr eated with micrococcal nuclease. The supernatant from the first 12,000 x g centr ifugation described abov e is transferred to an ultracentrifuge tube and centrifuged at 330,000 x g for 60 min at 4C. The pellet from this spin contains the cytoplasmic ribosomes and ribosome associated protein components, as well as the smooth and rough endoplasmic reticulum, micr osomes, exosomes, and other lipid related structures. The supernatant was removed and th e ribosome containing pellet was resuspended in 1.5 mL of hypotonic buffer. Resuspension of this pellet was facilitated by the use of a magnetic micro stir bar and stir plate at 4C, and typi cally required 30 min stir time. To standardize protein preparations, a 2 L a liquot was removed, diluted 1:250, and the absorbance at 260 nm was obtained. This roughly reflects the concentration of ribosomal RNA in the preparation, and readings in the range of 0.7-0.9 (175-225 A260 units undiluted) reflect the optimal concentration range. Preparations that exceed this range were diluted appropriately with hypotonic buffer until the desired absorbance is reached. Once an optimal A260 reading was attained, the total volume was measured and 1/7 volume of 4 M KCl was added. This addition raised the final KCl concentration to 0.5 M, disrupting the ionic and electrostatic intera ctions necessary for initiation factor association with the larger ribosomal comp lex. To allow this dissociation to proceed, the mixture was incubated for 15 min at 4C with stirring. The mixture was subsequently

PAGE 46

46 centrifuged again at 330,000 x g for 60 min at 4C. The resultant supernatant was removed and dialyzed against IF buffer [20 mM HEPES-KOH (pH=7.4), 120 mM K(CH3CO2), 5 mM Mg(CH3CO2)2, 5 mM DTT] for 2 h at 4C. The dialyz ed supernatant was divided into single use aliquots and stored at -80C. This dialyzed supe rnatant is defined as HeLa IF for the purposes of all experiments performed in the studies herein. HeLa S10 Translation-RNA Replication Reactions For all experim ents, extract preparations are thawed on ice immediatel y prior to their use, and aliquots are never reused on ce thawed. Additionally, transcript RNAs are stored in ethanol and are precipitated, washed, resuspended and qua ntitated immediately pr ior to their use. Transcript RNAs, once precipitated, were never reprecipitated and re used for later experiments. In general, all reaction components are thawed and stored on ice during experimental setup. Both translation and replicati on reactions utilized single us e aliquots of a 10X nucleotide reaction mix [155 mM HEPESKOH (pH=7.4), 600 mM K(CH3COOH), 300 mM creatine phosphate, 4 mg/mL creatine phosphokinase, 10 mM ATP, 2.5 mM GTP, 2.5 mM UTP]. This reaction mix includes optimal buffers and salts for PV translation and an ATP regenerating system, but excludes CTP. This omission allows for later use of [32P]CTP to radiolabel RNA replication products. RNA Programming and Translation HeLa S10 translation reactions were prepared by com bining 50% (by volume) HeLa S10 extract, 20% (by volume) HeLa IF, 10% (by volume) 10X nucleotide reaction mix, 2 mM guanidine hydrochloride (GuHCl), template RNA, and sterile/RNase-free water. Purified template RNA was precipitated, resuspended in sterile/RNase-free wa ter, and quantitated spectrophotometrically. Unless otherwise specified in an individual experimental methodology, 4 pmol of purified template RNA was used to program the HeLa S10 translation reactions. In

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47 cases where multiple RNAs were required (e.g. trans -replication experiments), 4 pmol total RNA was used. When RNA programmed translati on reactions were assembled, a 10 L aliquot was removed, to which 11 Ci (1 L) of L-[35S]-methionine (1,000 Ci/mmole; PerkinElmer) was added for metabolic labeling of newly synthesize d proteins. Both the HeLa S10 translation reactions and the [35S]-methionine labeling side reaction were incubated at 34C for 3-4 h. Following incubation, the HeLa S10 translatio n reactions were centrifuged to isolate pre-initiation replication complexes (PIRCs), wh ich is described below. Additionally, 5 L of the [35S]-methionine labeling side re action was added to 45 L of 1X Laemmli sample buffer (LSB; 112.5 mM TrisHCl (pH=6.8), 2% sodium dodecyl sulfate, 20% glycerol, 0.5% -mercaptoethanol, 0.02% bromophenol blue). This mixture was stored at -20C prior to analysis by SDS-PAGE. PIRC Isolation and RNA Replication Mem brane associated PV translation in the He La S10 translation reac tions resulted in the formation of replication complexes. The inclus ion of GuHCl in the translation reaction allowed these complexes to form, however initiation of negative-strand synthesis was blocked. These pre-fire complexes have been de fined as pre-initiation replica tion complexes (PIRCs). PIRCs can be obtained by centrifugation and isolation of the membrane pellet from HeLa S10 translation reactions. To do so, HeLa S10 tran slation reactions were centrifuged at 20,000 x g for 15 min at 4C. Supernatants were carefully removed so as not to disturb the membrane pellet. PIRC pellets were gently resuspended in replication buffer, which contained 50% (by volume) S10 buffer (40 mM HEPES-KOH (pH=7.4), 120 mM K(CH3CO2), 5.5 mM Mg(CH3CO2)2, 6 mM DTT, 10 mM KCl], 10% (by volume) 10X nucleotide reaction mix, 0.1 mg/mL puromycin, 5 M CTP, and 30 Ci [ -32P]CTP (800 Ci/mmol; PerkinElmer). Resuspension in a GuHCl-free buffer washed out the residual inhibitory GuHCl from the PIRCs,

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48 and replication was allowed to proceed by in cubation at 37C for 1 h. Following incubation, RNA replication reactions were stopped with 6 volumes of 0.5% SDS buffer, and were treated with 20 g of proteinase K for 15 min at 37C. Digested RNA replication reactions were subsequently extracted three times with phe nol:chloroform, extracted three times with chloroform, and precipitated by the addition of 3 volum es of 100% ethanol. Extracted product RNAs were stored in ethanol at -20C for at least 12 h, or un til analyzed by ge l electrophoresis. Analysis of Protein Sy nthesis by SDS-PAGE Protein synthesis was analyzed by 9-18% grad ient sodium dodecyl sufate polyacrylamide gel electrophoresis (SDS-PAGE). To do this, a vertical 0.75 mm 9-18% gradient resolving gel was cast using a gradient maker, and a 4% stacki ng gel was cast above the gradient gel. The ratio of acrylamide to bis -acrylamide used was 29:1, and the standard Tris-glycine discontinuous buffer system was also used. Samples were electrophoresed at constant current until the bromophenol blue dye front had exited the bottom of the resolving gel. Completed gels were fixed in 40% methanol/10% acetic acid for at le ast 15 min and rinsed with deionized water. Fixed gels were then impregnated with Am plify Fluorographic Reagent (GE Healthcare) by soaking for 10 min. Residual Amplify was rinsed away using deionized water and gels were transferred to chromatography paper for drying. Autoradiography was performed by exposure of dried gels to Kodak X-omat Blue XB-1 scientific imaging film at either -20C or -80C. Where applicable, quantitation of protein pro ducts was performed by phosphorimager using ImageQuant software (Molecular Dynamics). Analysis of RNA Replicat ion by Denaturing CH3HgOH Gel Electrophoresis Due to the uniquely elevated stability of an extended RNA-RNA duplex, complete denaturation of product RNA, particularly nega tive-strand RNA in a replicative form duplex, requires a powerful denaturant. For th is reason, methyl mercury hydroxide (CH3HgOH) agarose

PAGE 49

49 gel electrophoresis was utili zed to resolve and visualize RNA products from HeLa S10 translation-RNA replication reactions. Purified product RNA was recovered from ethanol precipitation by centrifugation, washi ng, and resuspension in 15 L of sterile/RNase-free water. An equal volume of CH3HgOH sample buffer [50 mM H3BO3/5 mM Na2B2O7 (pH=8.2), 10 mM Na2SO4, 1 mM EDTA, 25% glycerol, 0.05% bromophenol blue, 50 mM CH3HgOH] was then added to the resuspended replication product, an d allowed to denature for 5-15 min at room temperature. Denatured RNA pr oducts were resolved on a vertical 1% Seakem LE agarose gel which contained 5 mM CH3HgOH. Electrophoresis was performed at 70 mA constant current in 1X CH3HgOH running buffer [50 mM H3BO3/5 mM Na2B2O7 (pH=8.2), 10 mM Na2SO4, 1 mM EDTA]. For the first hour of electrophoresis, the buffer in the upper and lower buffer chambers were recirculated using a pe ristaltic pump to avoid depl etion of buffering capacity. Electrophoresis was halted when the bromopheno l blue dye front reached 1-2 cm from the bottom of the gel (typically 2.5 h total time). Gels were stained with 1.0 mg/mL ethidium bromide in 0.5 M NH4(CH3COOH) for 10 min and visualized on a UV transilluminator to ascertain equal loading/r ecovery. Gels were subsequently transferred to chromatography paper for drying. Autoradiography was performed by exposure of dried gels to Kodak X-omat Blue XB-1 scientific imaging film at -80C using a Biomax intensifying screen. Quantitation of RNA products was performed by phosphorimager using Im ageQuant software (Molecular Dynamics). Bacterial Protein Expression Bacterial protein expression plasm ids were ma intained as DNA stocks in TE at -20C and were only transformed into expression cells immediately prior to protein expression. Plasmid DNA was transformed into BL21(DE3) pLysS competent cells (Novagen) and a single colony was used to inoculate a 5.0 mL LB broth cult ure containing 50 g/mL ampicillin and 34 g/mL chloramphenicol. This culture was incubated ov ernight at 37C with shaking. A fresh 50 mL

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50 LB broth culture without antibioti c was then inoculated with 50 L of the overnight culture and incubated at 25C with shaking. Protein expres sion was induced for 2 h with 1 mM IPTG when the culture reached an OD600 of ~0.5. Cleared protein extracts were prepared as described in Andino et al (12). Briefly, cells were harvested, we ighed, washed once with phosphate buffered saline, and resuspended using 5.0 mL/g wet weight in lysis buffer (10 mM HEPES-KOH (pH=7.9), 20 mM KCl, 25 mM EDTA, 5 mM DTT, 1% Triton X-100). The resuspended cell pellet was frozen and thawed, sonicated for 30 s, and centrifuged at 150,000 x g for 15 min. The supernatant from this centrifugation was supplemen ted with glycerol to a final concentration of 20%, aliquotted and stored at -80C. Electrophoretic Mobility Shift Assays Riboprobe Synthesis DNA te mplates containing the cDNA for the PV 5CL were digested with Hga I, which cuts 30-nts past the 5CL. Cut DNAs were pur ified by phenol:chloroform extraction and ethanol precipitation. Radiolabeled 5CL probe s were made by T7 transcription of Hga I cut DNA template as described above for ribozyme optimi zed transcription, with one exception. Rather than 500 M CTP, a combination of 115 M non-labeled CTP and 4 Ci/L [ -32P]CTP (400 Ci/mmol) was used. Probes were purified dir ectly from the transcription reaction by passage over NucAway Spin Columns (Ambion), followed by a single phenol:chloroform extraction, single chloroform extraction, and ethanol precipita tion. Riboprobes were st ored in ethanol at -20C until immediately prior to th eir use, and were only reprecip itated for a maximum of two additional experiments. For quantitation of precipitated radiolabeled riboprobes, TCA precipitation, filtering, and scintillation c ounting was performed. Calculations were subsequently performed based on the specific activity of the [32P]CTP in the transcription reaction and the number of C residues in the given riboprobe.

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51 Binding Reactions and Gel Electrophoresis Electrophoretic m obility shif t assays (EMSA) were perf ormed based on a modified protocol described previously ( 13). Radiolabeled ri boprobes were prepared as described above, and protein was either obtained from HeLa S10 translation reactions or as recombinant protein from clarified bacterial lysa te. Binding reactions were performed by pre-incubating 1.5 L of HeLa S10 translation reaction mixture or 0.5-3 L of bacterial extract in a 9 L reaction, containing binding buffer [5 mM HEPE S-KOH (pH=7.9), 25 mM KCl, 2 mM MgCl2, 3.8% glycerol, 1.5 mM ATP, 20 mM DTT], 20 g yeast tRNA, and 40 U RNasin (Promega) at 30C for 10 min. To this preincubation mix, 20 fmol of 32P-labeled riboprobe was added to the reaction. The final binding reac tion was incubated at 30C for an additional 10 min prior to addition of 2 L loading buffer [0.1% bromophenol blue, 50% glycerol]. A 5% polyacrylamide [40:1 acrylamide:bisacrylamide] native gel contai ning 5% glycerol was ca st in 0.5X TBE buffer [176 M TrisHCl, 176 M H3BO3, 2 mM EDTA]. Prior to loading, the gel was pre-run at 4C for 30 min at 30 mA with constant current using 0.5X TBE as the running buffer. Ribonucleoprotein complexes were resolved by el ectrophoresis at 4C at 220 V with constant voltage. Electrophoresis was halted when the br omophenol blue dye front reached 3-4 cm from the bottom of the gel. The final gel was tr ansferred to chromatography paper, dried, and visualized by autoradiography as described above.

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52 CHAPTER 3 POLY(C) BINDING PROTEIN IS REQUIRED FOR EFFICIENT INITIATION OF POLIOVIR US NEGATIVE-STRAND RNA SYNTHESIS Introduction The poly(C) binding proteins (P CBP; also called hnRNP E and CP) represent a fam ily of poly(rC/dC) binding proteins which include hnRNP K and PCBPs 1-4 (109, 121, 128, 133). In addition to their nucleic acid bind ing specificity, this protein family is characterized by the presence and positioning of three highly homologous hnRNP K H omology domains(KH domains) (80, 188). In the case of the PCBPs, the first and third KH domains contain the primary nucleic acid binding activ ity, although the second domain may enhance binding affinity and/or specificity (65, 67). It is thought that the structure of this domain is highly conserved, regardless of surrounding sequence context, acting as an independent cassette which can be evolutionarily tuned to a speci fic function. Although initially characterized as RNA binding proteins involved in pre-mRNA metabolism, mo re recent work has described an increasingly globalized set of essential cellular processes in which PCBPs participate. While there is some degree of overlap in the sequen ces bound by the PCBPs, the use of alternate bind ing partners together with modulation of bi nding specificity and affinity, re sults in an immense number of potential regulatory targets and functions. As yet, the most extensively studied fam ily members are hnRNP K, PCBP1, and PCBP2. Current work has firmly established the invol vement of the PCBP protein family in mRNA stabilization, transcrip tional regulation, translat ional control, and apoptotic program activation (reviewed by (129)). The mRNA s targeted by these proteins are diverse as well, including -globin, 15-lipoxygenase, collagen I, tyrosine hydroxylase, er ythropoietin and androgen receptor (49, 64, 154, 162, 191, 211, 217). In addition, more recent work identified over 150 mRNAs in a hematopoietic cell line that interact, in vivo with PCBP2 alone (208). A

PAGE 53

53 number of interacting proteins have also been identified, including AUF1, HuR, SRp20 and Poly(A) Binding Protein, as well as other members of the PCBP family (6, 28, 49, 77, 93, 108, 110). Given the abundance and multi-functional nature of these proteins, it is not surprising that multiple viruses, both RNA and DNA alike, have e volved to utilize these proteins during various stages of their replication. The ORF57 protein of Kaposis Sarcoma-associated herpesvirus (KSHV) has been shown to interact with PCBP1, and this complex is capable of stimulating the translation of specific cellular a nd viral genes (147). In the case of human papillomavirus (HPV), PCBP1 interacts with one of the capsid protein mRNAs and down-regulates its translation (60). Interestingly, recent work reve aled markedly decreased levels of PCBP1 in cervical epithelial cells transf ormed by HPV, and demonstrated a direct correlation between cellular PCBP1 levels and progression to cervical cancer (169). Hepatitis C virus (HCV) binds PCBP2 at both the 5 and 3 NTR of its ge nomic RNA (189, 200), however the role of these binding events is yet to be understood. Many members of the family Picornaviridae have also been shown to utilize the PCBPs for their replication, including hepatitis A virus (HAV), human rhinovirus 14 (HRV-14), coxackievirus B3 (CVB3) as well as poliovirus (PV). One functional commonality is the requirement for PCBP2 in the cap-independent initiation of translation mediated by a type-I IRES (38, 66). Interestingly, although HAV does not have a classic type I IRES, PCBP2 is still utilized for translation initiation via interaction with an alternative sequence element in the 5 NTR (83). Even the type-II IRES of Encepha lomyocarditis Virus (EMCV), which does not require PCBP, will still compete for PCBP binding (66). This suggests a more generalized function for the PCBPs in picorn aviral translation initiation.

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54 Poliovirus (PV), in addition to the IRES, possesses a 5-terminal cloverleaf (5CL) structure that is essential for RNA replica tion and is conserved among all members of the Enterovirus genus (12, 13, 26, 93, 126, 196, 213, 219). The 5 CL is divided into four domains: stem a and stem-loops b, c and d (Figure 3-1A). Stem-loop d binds viral protein 3CDpro, and stem-loop b binds PCBP1 or PCBP2 (12, 13, 158). P CBP1/2 will bind to the 5CL in the absence of viral proteins, however the concomitant binding of 3CDpro results in a nearly 100-fold increase in PCBP binding affinity (79). While both the first and thir d KH domains bind poly(C) RNA with similar affinity, only the first KH domai n of PCBP1/2 is required to bind to the 5CL (65, 185, 209). The binding of PCBP1/2 and 3CDpro to the 5 cloverleaf is believed to play an important role in viral RNA replication (12, 13, 79, 213), and in RNA stability (144). In addition, PV RNA replication is inhibited in Poly(C)-depleted HeLa S10 extracts, strongly suggesting that PCBP binding to th e 5 cloverleaf is required in one or more steps of the viral RNA replication cycle (209). In the current study, we investigated the role of the PCBP-5CL RNP complex in PV RNA replication. Herein, we present data that demonstrates that the binding of PCBP to the 5CL is required to form the replication complex used to initiate PV negative-strand RNA synthesis. Furthermore, we describe the novel application of a protein-RNA tethering system in the functional analysis of essential cellular protein involve ment in virus replication. Using this system, we were able to overcome the difficulties presented in performing experiments involving RNAi, gene knockout, protein depletion or dominant negative inhibition of multi-functional, essential cellular proteins, such as the PCBPs. Moreover, we de monstrate the ability of this system to directly analyze and modify domains of a cellular protein, specifical ly as it pertains to virus replication.

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55 Results A Mutation in Stem-loop b of the 5 Clov erlea f Inhibits Negativ e-strand Synthesis To clarify the role of PCBP binding to th e 5CL in PV RNA replication, we used a subgenomic PV RNA transcript [P23 RNA] which encodes all of the essen tial viral replication proteins and forms functional RNA replication complexes in cellfree reactions. We compared the replication of wild-type P 23 RNA with the replication of the same RNA with a C24A mutation in stem-loop b of the 5 CL [P23-5CLC24A RNA] (Figure 3-1B). PV RNA replication was assayed using preinitiation RNA replication complexes (PI RCs) isolated from HeLa S10 translation-replication reactions as described in Chapter 2. To assay for negative-strand synthesis, we utilized P23 RNA transcripts wh ich contain two 5 term inal non-viral Gs that inhibit positive-stra nd initiation (25, 141). To first examine the ability of the 5CLC24A to bind PCBP, we pe rformed electrophoretic mobility shift assays (EMSA) using radiolabeled 5CL riboprobes containing either a wild-type or C24A stem-loop b. The C24A mutation has been previous ly shown to inhibit the formation of the essential 5 RNP complex (12, 77, 144, 158), and as expected, the PCBP-5CL RNP complex (complex I) was observed using 5CLWT RNA probe but not on 5CLC24A RNA probe. This demonstrated that PCBP2 does not bind to 5CLC24A RNA, as predicted. In RNA replication reactions containing P23-5CLC24A RNA, negative-strand synthesis was 10-20% of the amount observed with wild-type P23 RNA (Figure 3-2B). Additional work from our lab has shown that there is no defect in positiv e-strand synthesis of P23-5CLC24A RNA, despite the decreased levels of negative-strand synthesis (Sharma et al ., unpublished results). Taken together, these experiments demonstrated that P23-5CLC24A RNA was defective for negativebut not positive-strand synthesis

PAGE 56

56 (MS2)2 Protein-RNA Tethering System Tethered function assays have been used to st udy the activity of cellu lar proteins in mRNA metabolism and regulation apart from their RNA binding affinity and specificity (reviewed in 57). One system that is used for this type of assay takes advantag e of the high-affinity interaction of the MS2 bacteriophage coat prot ein with its cognate R NA stem-loop structure (47, 58). The MS2 tethered function system requires th e generation of an in-f rame fusion of the MS2 coat protein with the protein of interest. At the same time, the native protein binding site in the target RNA is replaced with the MS2 RNA stem -loop structure (47). Co-expression of the MS2 fusion protein targets the protein of interest to the MS2 stem-loop structure in the target RNA. This system was used by Kong et al to demonstrate that human -globin mRNA is stabilized by tethering an isoform of murine PCBP2 (murPC BP2-KL) (112). Because the MS2 coat protein binds to the MS2 stem-loop as a dimer, Hook et al examined the use of a covalent dimer of the MS2 protein, first described by Peabody and Lim ( 96, 163). This head-to-tail covalent dimer of MS2 coat proteins, here termed (MS2)2, results from the in-frame fusion of tandem MS2 open reading frames by a linker sequence. Using this approach, Hook et al. obs erved an increase in specificity and efficacy of the covalent dimer syst em, relative to that of a single MS2 fusion (96). For these reasons, we developed a protei n-RNA tethering system using the (MS2)2 covalent dimer to directly examine the role of PCBP2 tethered to the 5CL in PV RNA replication. In the case of wild-type PV RNA, PCBP1/2 binds to stem-loop b in the 5CL and viral protein 3CD binds to stem-loop d (Figure 3-3A). By replacing the majority of stem-loop b with an equally sized MS2 stem-loop structure [5CLMS2] (Figure 3-1C), we removed the PCBP binding site, thereby preventing endoge nous PCBP from binding to the 5CLMS2. In addition, expression of the (MS2)2PCBP2 covalent dimer fusion protei n, in the presence of the 5CLMS2 RNA, should tether PCBP2 to the 5CLMS2 via the interaction of the (MS2)2 covalent dimer with

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57 the MS2 RNA stem-loop structure (Figure 3-3B). By extension, we predict that PV RNA transcripts containing the 5CLMS2 will be defective for negative-strand synthesis, and that co-expression of the (MS2)2PCBP2 fusion protein in the same reaction should restore negative-strand synthesis definitively showing that PCBP2 is required for negative-strand initiation. (MS2)2PCBP2 Binds Specifically to 5CLMS2 RNA To establish the functionality of the MS 2 protein-RNA tethering system, we first performed electrophoretic mobility shift assays (E MSA) to examine the protein binding profile of the 5CLMS2 relative to that of the 5CLWT. As expected, the 5CLWT RNA probe formed a previously characterized PCBP-5CL RNP complex (complex I) with either endogenous PCBPs in HeLa S10 extracts or recombinant PCBP2 (rPCBP2) (Figure 3-4A, lanes 1-4) (12, 77, 158). In contrast, complex I was not formed in identical binding assays containing the 5CLMS2 RNA probe (Figure 3-4A, lanes 5-8). This dem onstrated that PCBP2 does not bind to 5CLMS2 RNA probe, as predicted. HeLa S10 translation-replica tion reactions were programme d with a non-translating RNA (mock translation) or protein expression RNAs which encoded either PCBP2, (MS2)2 or (MS2)2PCBP2. As expected, the PCBP-5CL complex (complex I) was formed in binding assays containing the 5CLWT RNA probe (Figure 3-4B, lanes 25) but not in assays containing the 5CLMS2 RNA probe (Figure 3-4B, lanes 7-10). The expression of exogenous PCBP2, (MS2)2 or (MS2)2PCBP2 had no significant effect on the formation of complex I with the 5CLWT probe (Figure 3-4B, lanes 3-5). However, the expression of either (MS2)2 or (MS2)2PCBP2 resulted in the formation of a new, slower-migrating RNP complex (complex II) with the 5CLMS2 RNA probe (Figure 3-4B, lanes 9-10) This demonstrated that the (MS2)2PCBP2 fusion protein binds specifically to 5CLMS2 RNA and not wild-type RNA.

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58 (MS2)2PCBP2 Restores Negative-strand Synthesis on P23-5CLMS2 RNA To ascertain the effectiveness of the MS2 pr otein-RNA tethering system, we measured the levels of negative-strand synt hesis observed with P23-5CLMS2 RNA in presence or absence of (MS2)2PCBP2. Negative-strand synthesis was measur ed in PIRCs isolated from HeLa S10 reactions that contained e ither P23 RNA or P23-5CLMS2 RNA and an equimolar amount of a protein expression RNA which expressed either (MS2)2, PCBP2 or (MS2)2PCBP2. In the reactions that contained P23 R NA, the co-expression of (MS2)2 or (MS2)2PCBP2 had little effect on negative-strand synthesis (Figure 3-5, lanes 1 & 3). There was a detectable increase in negative-strand synthesis in the presence of e xogenous PCBP2 (Figure 3-5, lane 2), which again is indicative of PCBP2 involveme nt in negative-strand synthesis. In the reactions containing P23-5CLMS2 RNA, negative-strand synthesis was reduced to barely detectable levels in the reactions that contai ned either the (MS2)2 or PCBP2 expression RNAs (Figure 3-5, lanes 4-5). In contrast, a large increase in negative-stra nd synthesis was observed in the reaction containing the (MS2)2PCBP2 expression RNA (Figure 3-5, lane 6). In this reac tion, negative-strand synthesis increased approximately 100-fold over the levels observed in the (MS2)2 or PCBP2 control reactions (Figure 3-5, compare lane 6 w ith 4 & 5). Therefore, these results clearly established the effectiveness of the MS2 protein-RNA tethering system and showed that PCBP2, either directly bound or tethered to the 5CL is required for effici ent initiation of PV negative-strand RNA synthesis. Deletion Analysis of PCBP2 Using the (MS2)2 Protein-RNA Tethering System To define the region of PCBP2 involved in the protein-protei n or protein-RNA interactions relevant to the initiation of negative-strand synthesis, we performed a deletion analysis of PCBP2, followed by a test of f unction using the MS2 protein-RNA tethering system. The PCBP2 coding sequence was divided into three re gions, each region containing one of the three

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59 conserved KH domains (Figure 3-6A). The coding sequence for each of these regions was expressed as an (MS2)2 fusion protein in reac tions containing P23-5CLMS2 RNA. As before, significant levels of negative-strand RNA s ynthesis was observed in the presence of (MS2)2PCBP2 compared to those observed in the (MS2)2 and PCBP2 reactions (Figure 3-6B, lanes 1-3). Neither e xpression of the (MS2)2KH1[Region] nor the (MS2)2KH2[Region] fusion proteins were able to suppor t negative-strand synthesis above background levels (Figure 3-6B, lanes 4-5). In contrast, the expression of the (MS2)2KH3[Region] fusion protein restored negative-strand synthesis to levels sligh tly higher than those observed with (MS2)2PCBP2 (Figure 3-6B, lanes 1 & 6). This suggested that the dominant domain in PCB2 that is required for negative-strand initiation resides in the C-terminal 130 amino acids of PCBP2, which includes the KH3 domain. A recent structural analysis of the KH doma ins of PCBP2 revealed an intramolecular interaction between the KH1 and KH2 domains that was predicted to influence the function of one or both of the domains (67). In consider ation of this potential effect on the functional activity, the amino terminal region of PCBP2, including both KH1 and KH2 domains, was fused to (MS2)2 [KH1/2[Region]], and the replication of P23-5CLMS2 RNA was measured in the presence of this fusion protein. In this reacti on, negative-strand synthesis was restored to about 70-80% of the levels observed with (MS2)2PCBP2 and about 50-60% of the levels observed with (MS2)2KH3[Region] (Figure 3-6C, lanes 1, 4 & 5). Th erefore, the results of this replication assay were consistent with the results of the structural studi es which predicted a functional relevancy for the intramolecular interaction be tween the individual KH1 and KH2 domains of PCBP2 (67).

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60 To ensure that the differences in replicati on efficiency observed in Figure 3-6 were not a secondary result of variations in the synthesis (or stability) of the (MS2)2 fusion proteins or the viral replication proteins, we measured the am ount of protein synthesi zed in each reaction. Labeled proteins were synthesized in He La S10 translation re actions containing [35S]-methionine and were examined by SDS-PAGE and autoradi ography (Figure 3-7). The results of this experiment indicated that all of the (MS2)2 fusion proteins were synthesized in similar amounts as full-length, intact proteins, and similar levels of the labeled viral proteins were synthesized in each reaction (Fig. 7). Taken together, these resu lts indicated that the observed differences in negative-strand synthesis that were a direct result of the efficacy of the given (MS2)2 fusion protein and not a result of differe nces in the levels of protein synthesis or protein stability. The Conserved KH3 Domain is Sufficien t to Support Negative-strand Synthesis Since the C-term inal KH3 containing fr agment supported the highest levels of negative-strand synthesis, we chose to analyze th is region further. To determine if the KH3 domain itself was responsible for the observed ac tivity, the residual Nand C-terminal amino acid sequences outside of the KH3 domain we re deleted to form the KH3[Domain] fusion protein construct (Figure 3-8A). Separate Nand C-terminal deleti ons were also made within the KH3 domain, removing the N-terminal -strand and the C-terminal -helix respectively (Figure 3-8A). Due to the structurally conserved na ture of the KH domain, these deletions would be expected to perturb the tertiary structure of the KH3 domain and to disrupt structurally dependent protein interaction surfaces. The coding seque nces for each of these mutants was fused to (MS2)2, and the replication of P23-5CLMS2 RNA was measured in the presence of the individual fusion proteins. Similar levels of protein synthe sis were verified as be fore by SDS-PAGE (data not shown). As expected, expression of the (MS2)2PCBP2 and (MS2)2KH3[Region] fusion proteins supported significant leve ls of negative-strand synthesis. Removal of the amino acids

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61 flanking the KH3 domain had no inhibitory e ffect on levels of negative-strand synthesis observed (Figure 3-8B, lanes 3 & 4), however, deletions in the KH3 domain itself (i.e. KH3 1 and KH3 3) completely inhibited negative-strand synthesis (Figure 3-8B, lanes 5 & 6). Therefore, the intact KH3 domain was requi red to support high levels of negative-strand synthesis. To confirm that the observed differences in negative-strand synthesis were not due to an unexpected change in a given (MS2)2 fusion proteins ability to bind to 5CLMS2 RNA, an EMSA was performed using 5CLMS2 RNA probe and each of the PCBP2 fragment (MS2)2 fusion proteins (Figure 3-9A). In each case, the labele d probe was shifted to form a slower migrating RNP complex similar to complex II in Figure 34B. We also examined the expression and integrity of each individual (MS2)2 fusion proteins by SDS-PAGE and autoradiography (Figure 3-9B). The results of these experi ments clearly showed that the (MS2)2 protein acts as a functional cassette to efficiently tether a fusion pr otein to the 5CL, regardless of that proteins identity. Additionally, all (MS2)2 fusion proteins appear to be synthesized in the similar amounts as stable, full-length proteins. Therefore, these results demonstrate that, wh en tethered to the RNA, the KH3 domain alone was sufficient to support init iation of PV negative-strand synt hesis. This activity was not an artifact of increased protei n concentration or binding affinit y, and from deletion experiments, this activity appears to be dependent on the intact tertiary structure of the KH domain. The Combined KH1-KH2 Domain Fragment Do es Not Utiliz e PCBP Dimerization to Promote Negative-strand Synthesis PCBP2 dimerization has been shown to be re quired for PCBP2s f unction in PV IRES translation, and an approximate dimerization domain within the KH2 domain was identified (29). This intermolecular dimerization could potentially be influenced by the previously discussed

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62 intramolecular interaction between the KH1 and KH2 domains (67). In th is case, it is possible that the (MS2)2KH1/2 fusion protein, when tethered to the RNA, forms heterodimers with endogenous full-length PCBP2 in the HeLa extrac ts. If so, the negative-strand synthesis observed in RNA replication reactions could be a result of the functi on of this additional molecule of PCBP, rather than a direct function of the KH1/2 domains. To determine if multimerization with endoge nous PCBP was responsible for the observed activity of the KH1/2 fragment, we deleted the 23 amino acid multimerization domain (MD; amino acids 125-158 of PCBP2) from the KH2 domain, in the context of the (MS2)2KH1/2 fragment [(MS2)2KH1/2MD] (Figure 3-10A) (29). Again, due to the structurally conserved nature of the KH domain, this deletion would be expected to significantly perturb the tertiary structure of the KH2 domai n. Replication of P23-5CLMS2 RNA was measured in the presence of the (MS2)2PCBP2, (MS2)2, (MS2)2KH1/2[Region] and (MS2)2KH1/2MD fusion proteins. Similar levels of protein synthe sis were verified as before by SDS-PAGE (data not shown). As expected, expression of the (MS2)2PCBP2 and (MS2)2KH1/2[Region] fusion proteins supported relative levels of negative-stra nd synthesis comparable to prev ious experiments (Figure 3-10B, lanes 1 & 3). Surprisingly, dele tion of the MD did not abolish the ability of the KH1/2 fusion protein to promote negative-strand synthesis, although the level of R NA product observed were decreased slightly. Therefore, the intact KH1/2 region, when te thered to the RNA, does not require multimerization to promote negative-strand synthesis. Multiple PCBP Isoforms Support Initia tion of Negativestrand RNA Synthesis Given the high degree of sequence conserva tion among members of the PCBP family, particularly within the KH domains, it was like ly that multiple PCBP isoforms may share the ability to promote PV negativestrand initiation. Additionally, it was already known that PCBP1 could bind to the 5CL and could also restore PV replication in Poly(C)-depleted cell extracts

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63 (77, 209). Further, the dominant splice vari ant of PCBP2, PCBP2-KL, only differs by the exclusion of 31 amino acids encoded by exon 8a, suggesting that PCBP2-KL would likely function similarly (Figure 3-11A) (76, 128). Together, PCBP1, PCBP2, PCBP2-KL represent the most closely related and highly abundant memb ers of this protein family (48, 127), and we would therefore predict that each of these isof orms would function at some level to restore negative-strand RNA synt hesis in the (MS2)2 tethering system. To determine the efficiency with which PCBP1 could function in this system, we measured the levels of negative-strand synthesis of P23-5CLMS2 RNA in the presence of either PCBP1, (MS2)2PCBP1, PCBP2 or (MS2)2PCBP2. As before, negative-strand synthesis was measured in PIRCs using equimolar amounts of P23-5CLMS2 RNA and the individua l protein expression RNA. In the reactions that contained P23-5CLMS2 RNA, co-expression of PCBP1 or PCBP2 alone was unable to promote negative-strand synthesis (Figure 3-11B, lanes 1 & 3). As predicted, (MS2)2PCBP1 was able support negative-RNA synthesis, and interestingly, the levels of negative-strand synthesis were slightly higher (~1.5-fold) than those observed in reactions containing (MS2)2PCBP2 (Figure 3-11B, lanes 2 & 4). To determine the efficiency with whic h PCBP2-KL could function in the (MS2)2 tethering system, we measured the levels of negative-strand synthesis of P23-5CLMS2 RNA in the presence of either PCBP2, (MS2)2PCBP2, PCBP2-KL or (MS2)2PCBP2-KL. Here again, negative-strand synthesis was measured in PIRCs using equimolar amounts of P23-5CLMS2 RNA and the individual protein expression RNA. In re actions that contained P23-5CLMS2 RNA, co-expression of PCBP2 or PCBP2-KL alone was unable to promote negative-strand synthesis (Figure 3-11C, lanes 1 & 3). As expected, (MS2)2PCBP2-KL was able support levels

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64 of negative-RNA synthesis similar to thos e observed in reactions containing (MS2)2PCBP2 (Figure 3-11C, lanes 2 & 4). Not All PCBP Family Members S upport Negative-strand synthesis Although their potential role in PV replication has neve r been investigated, the m ore distantly related members of the PCBP family (PCBP3, PCBP4, PCBP4A, and hnRNP K) are all expressed to varying levels in different tissue types (128). More importantly, we were able to detect mRNA corresponding to each protein in HeLa cell total RNA by RT-PCR, indicating the presence of each of these less abundant isoforms in HeLa cell extracts. HnRNP K is the most distantly related to all other PCBPs, and unde rgoes significant nucleo-cytoplasmic shuttling (135), but is predominantly localized to the nuc leus except during cell-c ycle signaling (118). PCBP3 and PCBP4/4A exhibit cy toplasmic localization and app ear to be excluded from the nuclear compartment (48), indicating their ava ilability to participate in cytoplasmic PV replication. PCBP4A is a spli ce variant of PCBP4, differing only in the carboxy-terminal amino acids, but contains an identical KH1, KH2, vari able region, and 90% of KH3 (128). Despite a higher degree of amino acid similarity with in individual KH domains, PCBP3/4/4A are significantly divergent from PCBP1/2 and may not re tain all necessary func tions relative to PV replication (Figure 3-12A). To determine the relative abilities of the vari ous PCBP family member s to function in the (MS2)2 tethering system, we measured the levels of negative-strand synthesis of P23-5CLMS2 RNA in the presence of either (MS2)2, (MS2)2PCBP1, (MS2)2PCBP2, (MS2)2PCBP2-KL, (MS2)2PCBP3, (MS2)2PCBP4, (MS2)2PCBP4A, or (MS2)2hnRNP-K. As before, negative-strand synthesis was measured in PIRCs using equimolar amounts of P23-5CLMS2 RNA and the individual protein expression RNA. As before, (MS2)2PCBP1, (MS2)2PCBP2, or (MS2)2PCBP2-KL were all able to promote ne gative-strand RNA synthesis of P23-5CLMS2

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65 RNA to similar levels (Figur e 3-12, lanes 2-4). The (MS2)2 fusion of the most distantly related, predominantly nuclear hnRNP K was unable to support significant leve ls of negative-strand synthesis, as may have been expected (Figure 312, lane 8). Surprisingly, the distantly related PCBP4, and to a lesser extent P CBP4A, functioned better than P CBP1, 2, or 2KL, whereas the more closely related PCBP3 appeared to support very modest levels of negative-strand RNA synthesis (Figure 3-12, lanes 5-7). To further determine if these more distantly related PCBPs could plausibly be involved in natural PV replication, the ability of each of the isoforms wa s assayed for its ability to bind the wild-type 5CL. RNA binding was ascertained by EMSA using a 5CL riboprobe and bacterially recombinant PCPBs as described in Chapter 2. Since the splice variants PCBP2-KL and PCBP4A maintain the same RNA binding determinants as their parental proteins, only PCBP2 and PCBP4 were assayed. As previously obser ved, both PCBP1 and PCBP2 were able to form RNP complexes with the PV 5CL, and corresp onding with its significa nt divergence, no RNP complex was formed in the presence of hnRNP K (Figure 3-13, lanes 2, 3, & 6). Interestingly, both PCBP3 and PCBP4 were able to form RN P complexes with the PV 5CL (Figure 3-13, lanes 4 & 5), suggesting that th ese isoforms have th e potential to, if present, form an RNP complex with viral RNA and partic ipate in negative-strand initiation. Discussion The work presented here dem onstrated the re quirement for PCBP in the initiation of PV negative-strand synthesis. Furthe rmore, we established that a direct PCBP-RNA interaction was not required to mediate this func tion by developing and using the (MS2)2 protein-RNA tethering system to investigate PV negative-strand synthesis. We demonstrated the utility of this system in analyzing regions in an essentia l cellular protein, relative to PV replication, without affecting other viral and cellular processes in which the protein is involve d. In doing so, we have shown

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66 the KH3 domain of PCBP2, when tethered to the RNA, was able to support the initiation of negative-stand RNA synthesis. Th is suggests that a structurally conserved prot ein-protein or protein-RNA interaction surface which is required fo r negative-strand initiation exists within this conserved domain. We have also noted the functional redundancy of the combined KH1/2 domains of PCBP2 relative to PV RNA replicati on, consistent with rece nt work demonstrating the in vitro functionality of PCBP2 with the KH3 domain deleted (165). Prior Indications of PCBP Involve ment in Poliovirus RNA Replication Initial in viv o studies identified an RNP complex form ed at the 5 end of PV genomic RNA which appeared to be i nvolved at some stage of RNA replication (13). Further investigation of this complex revealed the pr esence of both a viral (3 CD) and cellular (PCBP2) protein, and showed that disrupti on of this complex inhibited RNA replication (12, 158). Further in vitro analysis using a PCBP binding site point muta nt (C24A) revealed an additional role for PCBP in PV RNA stability (144). Using an al ternative approach, Walte r et al. showed that replication of a dicistronic PV RNA replicon was inhibited in cell extracts which were depleted for PCBPs (209). However, the above work was e ither unable to directly account for effects on RNA stability or to differentiate between defect s in negativeand positive-strand synthesis. Our results showed that the C24A mutation, which inhibits PCBP binding, also inhibits negative-strand synthesis w ithout inhibiting positive-st rand synthesis. Using a trans -replication assay, we showed definitively that this defect was not a secondary effect of a deficiency in either protein synthesis or RNA stabilit y. Therefore, these findings s uggested but did not prove that PCBP is a co-factor in the initiati on of PV negative-strand synthesis.

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67 (MS2)2 Protein-RNA Tethering Assay Demonstrated that PCBP is Required for Poliovirus Negative-strand Synthesis We directly addressed the role of PCBP2 in the initiation of PV negative-strand synthesis, by developing a protein-RNA tethering system. This system utilized the high affinity interaction of MS2 bacteriophage coat prot ein with its cognate RNA stem-l oop (47, 58). We modified this system as described by Hook et al. to take a dvantage of the added specificity and efficacy conferred by using an MS2 pr otein covalent dimer, (MS2)2 (96). By replacing the natural PCBP binding site in the 5CL of PV geno mic RNA with the MS2 stem-loop (5CLMS2), we were able to target the (MS2)2PCBP2 fusion protein to the 5CLMS2 RNA. This system has many significant advantages ov er other approaches used to functionally characterize cellular protein involvement in virus replication. It is very difficult to isolate individual functions of multi-functional cellular pr oteins using techniques such as RNAi, gene knockout, protein depletion and dominant-negativ e inhibition, which can result in a broad spectrum of downstream effects unrelated to the f unction of interest. In addition, some of these techniques are not feasible in certain systems, while others present significant technical challenges. The (MS2)2 protein-RNA tethering syst em could be adapted for in vivo and in vitro use, and functional analysis with this system can be performed with minimal disruption of other normal cellular processes. In the PV life-cycle, PCBP2 is used in both IRES-dependent translation and RNA replication. Our analysis of PCBP2s role in replication using the (MS2)2 protein-RNA tethering system can be performe d without affecting PCBP2 binding to the IRES, since the fusion protein is targeted specifically to the 5CL. The system also permits us to precisely define the protein bound to the 5CL thereby providing a platform to perform mutagenic analysis of protein func tion in a straightforward manner.

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68 Using the (MS2)2 protein-RNA tethering system, we observed a significant defect in negative-strand synthesis of a 5CLMS2 template RNA, concurrent with the loss of PCBP binding to the 5CLMS2. We then demonstrated a restora tion of negative-strand synthesis upon co-expression of the (MS2)2PCBP2 protein. This conclusively showed that the presence of PCBP2 at the 5CL of PV genomic RNA is required for the initia tion of negative-strand synthesis. Furthermore, PCBPs function is not mediated through direct protein-RNA interaction with the 5CL, since tethered PCBP2 wa s capable of restoring RNA replication. The requirement for PCBP in negative-strand in itiation is consistent with the current model of PV replication complex formation i nvolving genome circularization mediated by protein-protein interactions be tween RNP complexes formed at the 5 and 3 ends of PV genomic RNA (26, 93, 126, 196). By this model, P CBP2 bound or tethered to the 5CL interacts with PABP bound to the poly(A) tail, there by circularizing the ge nome and allowing the subsequent initiation of negative-strand synthesis. The Combined KH1 & KH2 Fragment or th e KH3 Domain of PCBP2 is Required for Negative-strand Initiation An i mportant application of the tethered f unction assay is mutational analysis of the tethered protein to determine the regions involved in protein-protein interactions. This illustrates yet another advantage of this system in that functional analysis of PCBP fragments can be performed without requiring direct binding to the 5CL. Using th is system, we were able to show that the KH3 domain of PCBP2 contains sequences and structures sufficient for the functional interactions involved in the initiation of negative-strand RNA synthesis. Based on the current circularization model, the key proteinprotein interactions w ould be between PCBP, PABP and 3CD, however it is also possible that other proteins are involved, or that PCBP interacts with an as yet unknown RNA elem ent in the 3 end of the viral genome.

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69 We were also able to observe a functi onal redundancy in PCBP2 residing in the amino-terminal KH1 and KH2 domains that wa s capable of mediating similar critical interactions. This function was not observed wh en either the KH1 or KH2 domains were used separately, consistent with recent NMR structural data, which indicated structural differences between the PCBP2 KH1 or KH2 domains indivi dually and a tandem KH1/2 construct (67). Multiple studies from Du et al. have identified large hydrophobic faces on KH1 and KH2 which would allow the two domains to interact intramol ecularly, further intertwining the function of the KH1 and KH2 domains (67, 68). These results ar e also consistent with the recent work by Perera et al. which demonstrated the ability of PCBP 2 with a KH3 deletion to restore PV RNA replication in Poly(C)-depleted cell extracts (165). A dimeri zation domain in PCBP2 has been identified and it was shown that this sub-dom ain within KH2 was required for IRES function (29). This domain was deleted from the KH1/2 fusion protein and was s hown to be dispensable for negative-strand synthesis, indicating that th e activity of KH1/2 frag ment does not require multimerization. The fully functional KH3 fragment does not contain any established dimerization sequences; so here again, dimerization of PCBP2 does not appear to be required to mediate the interactions required for negative-strand initiatio n. This does not rule out the possibility that dimerization of PCBP may be involved in physio logic PCBP binding to the 5CL, since this interaction has been bypassed using the protein-RNA tethering system. A Subset of PCBP Isoforms Support Initia tion of Negative-strand R NA Synthesis The PCBPs represent a family of poly(rC/d C) binding proteins which include hnRNP K and PCBPs 1-4, and two additional predominant splice variants, PCBP2-KL and PCBP4A (109, 121, 128, 133). This protein family is characterized by the presence and positioning of three highly homologous KH domains (80, 188), which were originally characterized as RNA binding

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70 domains (65, 67) but have been evolutionarily retuned to perform a dditional or alternative functions. Given the high degree of sequence conservation among member s within the PCBP family, particularly between corresponding KH domains, it was likely that some functional overlap between different isoforms of PCBP. Sp ecifically, different PCBP isoforms could share the ability to promote PV negative-strand initiatio n. Both PCBP1 and PCBP2 are able to bind to the 5CL and were able restore PV replication in poly(C)-depleted cell extracts (77, 209). Since PCBP2 and its splice variant PCBP2-KL share the same RNA binding determinants and only differ by 31 amino acids, it is likely that PCBP2KL functions similarly (76, 128). Through the application of the (MS2)2 protein-RNA tethering system, we have shown that PCBP1, PCBP2, and PCBP2-KL all share similar functionality re lative to PV RNA replication and can support similar levels of negative-strand synthesis. Despite a higher degree of amino acid simila rity within individual KH domains, PCBP3, PCBP4, and PCBP4A are significantly divergen t from PCBP1/2/2KL and may not retain all necessary functions relative to PV replication. Even within th e KH domains, hnRNP K is clearly the most divergent and distantly related PCBP isoform, and woul d be predicted to share very little functional similarity with the other family members (128). We have shown by RT-PCR that the more distantly related and less abundant PCBP isoforms are in fact expressed in HeLa cells, and have further shown that PCBP3 and PCBP4/4A, but not hnRNP K, are capable of forming critical RNP complexes with the PV 5CL. Again, using the (MS2)2 tethering system, we have shown that PCBP4 and PCBP4A are capable of supporting higher levels of negative-strand synthesis than the levels supported by PCBP1/2/2KL. In terestingly, PCBP3 was only able to support minimal levels of negativ e-strand synthesis, suggesting that PV RNA replication may be inhibited in cells which express high levels of PCBP3. Taken together this

PAGE 71

71 suggests that during natural PV infection, PCBP 3, PCPB4 or PCBP4A would each possess the ability to bind to the 5CL and incorporate into the RNP complexes critical for initiation of negative-strand RNA synthesis. Furthermore, PCBP3 could potenti ally act as an inhibitor of viral RNA replication by forming a non-functiona l RNP complex with the 5CL of PV RNA.

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72 Figure 3-1. Diagrams of the wild-type and mutant 5 cloverleaf. A) The PV 5CL is divided into four domains: stem a, and stem-loops b, c and d. A region of stem-loop b functions as the PCBP binding site, whereas 3CDpro binds to structural elements in stem-loop d. B) The 5CLC24A contains a single mutation (indicat ed in red) in the PCBP binding site of stem-loop b. C) The 5CLMS2 has had the majority of stem-loop b replaced with the MS2 bacteriophage coat pr otein binding site (MS2 stem-loop).

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73 Figure 3-2. The C24A mutation inhibits PCBP binding and negative-st rand RNA synthesis. A) Electrophoretic mobility shift assay (EMSA) using radiolabeled RNA probes, either 5'CLWT RNA (lanes 1-3) or 5'CLC24A RNA (lanes 4-6). The RNA probe was either run alone (lanes 1 & 4), with He La S10 extracts (lanes 2 & 5), or with bacterially expressed rPCBP2 (lanes 3 & 6) The specific RNP complex formed with the 5'CLWT RNA probe and cellular PCBP is labeled as complex I. B) Replication of P23 RNA and P23-5CLC24A RNA was measured using PIRCs isolated from HeLa S10 reactions. Radiolabeled product RNA was visualized by denaturing CH3HgOH-agarose gel electrophoresis a nd autoradiography. These P23 RNA transcripts allow only negative-strand synt hesis due to the presence of two non-viral G residues at the 5 end as a result of T7 transcription.

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74 Figure 3-3. Schematic of the (MS2)2 protein-RNA tethering syst em. A) Schematic of the wild-type 5CL RNP complex. This comp lex consists of PCBP bound to stem-loop b and 3CDpro bound to stem-loop d. B) Schematic of the 5CLMS2 RNP complex. Endogenous PCBP in cell extracts (or recombinant PCBP) is no longer able to bind to the 5CL because stem-loop b has been replaced with the MS2 stem-loop. In the absence of PCBP binding, 3CDpro can still bind to the 5CLMS2, but at lower affinity. When the (MS2)2PCBP2 fusion protein is provide d, it is recruited to the 5CLMS2 via the (MS2)2 interaction with its cognate stem-loop. This effectively tethers PCBP2 to the 5CL, forming a surrogate 5CL RNP holocomplex.

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75 Figure 3-4. The 5CLMS2 binds (MS2)2 fusion proteins but does not bind PCBP2. A) Electrophoretic mobility shift assay (EMSA) using radiolabeled RNA probes, either 5'CLWT RNA (lanes 1-4) or 5'CLMS2 RNA (lanes 5-8). The RNA probe was either run alone (lanes 1 & 5), with HeLa S10 mock translation reactions (lanes 2 & 6), with bacterially expr essed rPCBP2 (lanes 3 & 7) or a vector control bacterial extract (lanes 4 & 8). The specific RNP complex formed with the 5'CLWT RNA probe and endogenous cellular PCBP is labeled as complex I. B) EMSA using either 5'CLWT RNA probe (lanes 1-5) or 5'CLMS2 RNA probe (lanes 6-10). The probe was either run alone (lanes 1 & 6), with a HeLa S10 mock translation reaction (lanes 2 & 7), or with HeLa S10 translation reacti ons in which the indicated proteins were expressed (lanes 3-5, 8-10). Specific RN P complexes were formed with the 5'CLWT RNA and endogenous cellular PCBP (c omplex I), or with the 5'CLMS2 RNA and (MS2)2 or (MS2)2PCBP2 (complex II).

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76 Figure 3-5. The (MS2)2PCBP2 fusion protein restores ne gative-strand synthesis of a 5CLMS2 RNA template. Negative-strand synthesi s was measured in reactions containing either P23 RNA (lanes 1-3) or P23-5'CLMS2 RNA (lanes 4-6) using PIRCs isolated from HeLa S10 reactions. Each reacti on contained either P23 RNA or P23-5CLMS2 RNA and an equimolar amount of a protei n expression RNA which expressed either (MS2)2, PCBP2 or (MS2)2PCBP2, as indicated. Both te mplate RNAs were capped to ensure equal template stability. Radiolabeled product RNA was visualized by denaturing CH3HgOH-agarose gel electrophoresis and autoradiography

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77 Figure 5-6. Identification of th e functional domains within PCBP2 that restore negative-strand RNA synthesis of a 5CLMS2 template RNA. A) Schematic of the domain structure of PCBP2, including the conserved KH1, KH2 a nd KH3 domains. Each PCBP2 region depicted was fused to (MS2)2 and assayed in replication reactions. B & C) Negative-strand synthesis was meas ured in reactions containing P23-5'CLMS2 RNA and an equimolar amount of an RNA wh ich expressed the indicated RNA. The total molar RNA concentration and molar RNA ratio were maintained in each reaction, and the input templa te RNA contained a 5 cap.

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78 Figure 3-7. Levels of protein synthesis observed in the (MS2)2 protein-RNA tethering replication reactions. HeLa S10 translat ion reactions which correlate to those described in Figure 5-6 were incubated with [35S]methionine to metabolically label all newly synthesized protein pr oducts. The labeled protei ns synthesized in these reactions were analyzed by SDS-PAGE and autoradiography. Each reaction contained an equimolar amount of P23-5CLMS2 RNA and the indicated (MS2)2 fusion protein expression RNA.

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79 Figure 3-8. Characterization of the KH3 domain using the (MS2)2 protein-RNA tethering system. A) Schematic of PCBP2 and the KH3 domain deletion mutants used in this experiment. B) Negative-strand synthesis was measured using PIRCs isolated from HeLa S10 reactions containing P23-5'CLMS2 RNA and an equimolar amount of a protein expression RNA as indicated above. The total mo lar RNA concentration and molar RNA ratio were maintained in each reaction, and the input template RNA contained a 5 cap.

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80 Figure 3-9. The (MS2)2 fusion proteins are evenly expr essed, stable, and bind to 5CLMS2 with similar affinity. A) An EMSA was performed using a radiolabeled 5CLMS2 RNA probe. The probe was either run alone (lan e 1), with a HeLa S10 mock translation reaction (lane 2), or with HeLa S10 tran slation reactions in which the indicated proteins were expressed (lanes 3-11). B) Portions of the same HeLa S10 translation reactions used above were incubated with [35S]methionine, and the labeled protein products were analyzed by SD S-PAGE and autoradiography.

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81 Figure 3-10. The ability of the combined KH1/2 domains to restore negative-strand synthesis does not require the multimerization domai n. A) Schematic of PCBP2, KH1/2 region, and the multimerization domain deletion mu tant used in this experiment. B) Negative-strand synthesis was measured using PIRCs isolated from HeLa S10 reactions containing P23-5'CLMS2 RNA and an equimolar amount of a protein expression RNA as indicated above. The total molar RNA concentration and molar RNA ratio were maintained in each reaction, and the input template RNA contained a 5 cap.

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82 Figure 3-11. PCBP1, PCBP2, and P CBP2-KL restore negative-strand synthesis to similar levels in the (MS2)2 protein-RNA tethering system. A) Schematic of the domain structure of PCBP1, PCBP2 and PCBP2-KL. Each P CBP isoform depicted, as well as its corresponding (MS2)2 fusion protein, was assayed in replication reactions. B & C) Negative-strand synthesis was meas ured in reactions containing P23-5'CLMS2 RNA and an equimolar amount of an RNA which expressed the indicated protein. The total molar RNA concentration and mo lar RNA ratio were maintained in each reaction, and the input templa te RNA contained a 5 cap.

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83 Figure 3-12. PCBP4/4A, but not PCBP3 or hnRNP K, restores ne gative-strand synthesis in the (MS2)2 protein-RNA tethering system. A) Sche matic of the domain structure of the PCBPfamily. Each PCBP isoform depicted was fused to (MS2)2 and assayed in replication reactions. B) Negative-stra nd synthesis was measured in reactions containing P23-5'CLMS2 RNA and an equimolar amount of an RNA which expressed the indicated protein. The total molar RNA concentration and molar RNA ratio were maintained in each reaction, and the input template RNA contained a 5 cap.

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84 Figure 3-13. All PCBP family proteins, except hnRNP-K, bind to the PV 5CL. A) An EMSA was performed using radiolabeled 5CLWT RNA probe and clarified bacterial recombinant protein extract. The RNA pr obe was incubated with a vector only expression control (lane 1) or with a bacterially expressed recombinant PCBP isoform as indicated (lanes 2 & 6). The specif ic RNP complexes formed with the 5'CLWT RNA probe and the various PCBP is isoforms are indicated as isotypes of complex I

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85 CHAPTER 4 2BC-P3 IS THE CRITICAL CIS-ACTING VIR AL PROTEIN PRECURSOR DIRECTING INITIATION OF POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS Introduction Each stage o f the viral life-cycle must be carefully orchestrated, both spatially and temporally, to optimize total virus yield. The evolutionary imperative to do so is met by a myriad of obstacles at every step, yet viruses have developed multiple mechanisms to overcome these challenges and replicate efficiently. One common theme among these adaptations is a close coupling between sequential st eps of viral replication; predicating the initiation of one step, not simply on completion of the previous step, but also on the spatial availability and functionality of the products of that previous step. The primary means by which many viruses accomplish this is through extensive rearrangement of the host cell archit ecture and the creation of structures known as virus incl usions or virus factor ies (reviewed by (145)). While the creation of these structures is well established, the mechanisms which drive the coupling of the viral life-cycle within these structures continue to be of great interest. Poliovirus infection has been shown to causes dramatic membrane rearrangements resulting in the formation of characteristic vesicl es within the host cell cy toplasm (44). For PV, the cytoplasmic surface of these membrane vesicl es serves as the site of viral replication complex assembly, RNA replication, and infec tious particle assembly (34, 35, 43-45, 51, 167). Recent data from Egger & Bienz suggest a tight coupling between vira l translation at the endoplasmic reticulum and the formation of thes e membrane vesicles as well as concurrent replication complex formation (70, 71). Furthe rmore, PV defective interfering particles all maintain the correct reading frame and harbor deletions within the capsid coding region, suggesting that active translati on and RNA replication are linked (85, 115). This coupling was further substantiated by Novak a nd Kirkegaard who showed that cis translation of mutant viral

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86 RNA is a prerequisite to the replication of that particular RNA (151). Additionally, RNA replication is functionally coupled to infecti ous particle assembly, such that only newly synthesized positive-strand virion RNA is encapsida ted efficiently (152). Taken together, these studies depict a highly organized and well coordinated process by which PV infection progresses, each step inextricably tied to the initiation of the next, in an effort to maximize replication efficiency and virus yield. While it is clear that PV does adhere to a tightly coupled re plication strategy, the various molecular mechanisms by which this coupling oc curs remain unclear. In an effort to characterize some of these mechanisms, we utilized the HeLa S10 translation-replication system to probe the relationship between viral translation and initiation of RNA replication. To do this, we performed trans -complementation of PV subgenomic RN As expressing discrete polyprotein precursors and assayed for the ab ility of these subgenomic RNAs to serve as templates for negative-strand RNA synthesis. He rein, we show that the coupling of translation to subsequent negative-strand synthesis is not si mply due to the act of transla tion itself, but the function of a specific gene product(s). Furthermor e, we identify the critical region(s) of the PV polyprotein as that which includes 2B and 3Dpol proteins and/or their precursors. This data, in combination with previous observations, strongly indi cates that it is the translati on of the 2BC-P3 precursor in cis that drives template selection for membrane associated negative-strand RNA synthesis. Results To determ ine if the marked coupling of tran slation and RNA repli cation observed during in vivo PV infection was recapitulated in cell-free rep lication reactions, we performed a series of trans complementation experiments utilizing the HeLa S10 translation-replication system. In all cases, two RNAs were present in these reactions : the template RNA, which acts as the RNA

PAGE 87

87 replication template, and the he lper RNA, which acts as the trans protein provider but does not itself act as a replication template. Efficient PV Negative-strand Synthesis Re quires Translation of Viral Template RNA We and others have established that neither the capsid proteins nor the capsid protein coding region is required for PV genom e translation and RNA replication both in vitro and in vivo (61, 85, 106, 115). W ith this in mind, the te mplate RNA used was either a subgenomic RNA which expressed all PV re plication proteins [P23 RNA] or a similar RNA containing a frameshift mutation and subsequent early termin ation codons [FS23 RNA] (Figure 4-1A). In both cases, a full-length PV helper RNA was include d in the reaction to pr ovide all PV proteins and polyprotein precursors in trans This full-length helper RNA contains a five nucleotide deletion in the 3 NTR which has been previously shown to inhibit ne gative-strand synthesis without affecting translation [PV1 GUA3 RNA] (26). In all cases, detected product RNA represents only negative-strand synthesis as a result of two non-viral G residues at the 5end of all template RNAs used in this study. PV negative-strand RNA synthesis was assaye d using preinitiation replication complexes (PIRCs) isolated from HeLa S10 translation-rep lication reactions as previously described in Chapter 2. Equimolar amounts of template RNA [P23 or FS23] and helper RNA [PV1 GUA3] were co-translated and subsequent nega tive-strand synthesis was measured by [ -32P]CTP incorporation and visualized by denaturing CH3HgOH-agarose gel electrophoresis and autoradiography. As shown in Figure 4-1B, nega tive-strand synthesis from the non-translating FS23 template RNA was reduced 5 to 10-fold rela tive to that observed from the P23 template RNA, which translated all replication proteins in cis As expected, there were slightly increased levels of the viral P23 proteins produced in the reaction containing both P23 and PV1 GUA3

PAGE 88

88 RNAs relative to that produced in the reac tion containing the non-tr anslating FS23 RNA. However, in reactions in which the amount of helper RNA was doubled to approximate reaction conditions with elevated replicat ion proteins, the levels of nega tive-strand synthesis in reactions containing FS23 RNA remained significantly lower than those observed with P23 RNA (data not shown). Additionally, if proteins coming in cis and in trans contributed equally to RNA replication, one would expect th e levels of negative-strand sy nthesis supported by proteins coming only in trans to be half of that observed wh en proteins are provided both in cis and in trans rather than the 10-15% that was experiment ally determined (Figure 4-1B, compare lanes 1 & 2). Therefore, these data indicate that th e observed difference in negative-strand synthesis between P23 and FS23 RNA templates was predomin antly due to the translation status of the RNA being replicated. In many translation systems, RNAs harboring premature termination codons or RNAs that are translationally inactivated ar e often subject to nonsense medi ated decay or other forms of RNA degradation (56, 146). To determine if th e observed negative-strand synthesis defect of FS23 was an indirect effect of RNA instabil ity we performed RNA stability assays under conditions identical to those used to assay for negative-strand RNA synthesis. Radiolabeled input RNA, either P23 or FS23 RN A, was co-translated with PV1 GUA3 RNA and the amount of input RNA remaining was a ssessed at various times by CH3HgOH-agarose gel electrophoresis and autoradiography. There was no detectible defect in the stability of FS23 input RNA relative to P23 RNA (Figure 4-1C, compare lanes 1-4 to lanes 5-8), despite premature translation termination on FS23 RNA. This result confir med that the difference in the levels of negative-strand synthesis observed between P 23 and FS23 RNA templates was not due to a secondary stability defect arising from the absence of elongating ribosomes.

PAGE 89

89 Template RNA Translation Alone is Not Sufficient to Promote Efficient PV Negative-strand RNA Synthesis To determ ine if translation of an entire read ing frame and/or translation termination in the authentic context was required for efficient in itiation of negative-str and synthesis, a PV1 subgenomic RNA was constructed in which th e sequences coding for 2A through 3C and a portion of 3D were deleted (P1-3D* RNA; Figure 4-2A). This RNA is actively translated to produce a fusion of the P1 capsid precursor protei n and a carboxy terminal fragment of 3D, but does not produce any active 3Dpol, and terminates translation in the authentic context. A derivative of this subgenomic RNA was then co nstructed by the addition of a frameshift mutation at nucleotide 1119 of P1-3D* RNA (FS1-3 D* RNA; Figure 4-2A). Translation of this RNA initiates properly but terminates prematurely, resulting in synthesis of a truncated protein product and incomplete ribosomal transit of the template RNA. Negative-strand RNA synthesis wa s assayed as described above in reactions containing either P1-3D* or FS1-3D* template RNA and an equimolar amount of PV1 GUA3 helper RNA. As shown in Figure 4-2B, no significant difference in the levels of nega tive-strand synthesis was observed between reactions containing P1-3D* or FS1-3D* RNA. Additionally, the amount of negative-strand synthesis observed from both reac tions is significantly reduced from those observed from P23 RNA which translates all viral replica tion proteins in cis (data not shown). These data clearly show that neither complete ribosome translocation through a template RNA, nor termination of translation in the authenti c context, is sufficient to direct efficient negative-strand synthesis from that template. Furthermore, this result strongly suggests that the previously observed cis -translation enhancement of negative-strand synthesis is primarily due to the activity of a viral translation product in cis

PAGE 90

90 Translation through the 3D Coding Region in cis is Necessary for Efficient PV Negative-strand Synthesis To determ ine which viral protein pro duct or products were required in cis for efficient PV negative-strand synthesis, a series of subgenomic template RNAs was constructed based on the P23 RNA used above. For each construct, two stop codons were inserted after the terminal amino acid residue of the desired protein within the context of the en tire polyprotein coding region of P23 RNA (Figure 4-3A). These construc ts all maintained the identical RNA sequences and structures (with the exception of the stop cod ons) as the parent P23 RNA, but translated only a defined amino-terminal portion of the PV polypr otein. Translation of these RNAs initiated with 2A and progressed normally until reachi ng the inserted stop codons, whereby P23-2ASTOP translated 2A, P23-2BSTOP translated 2AB, P23-2CSTOP translated 2ABC, and so forth (Figure 4-3A). In all reactions, the full-length PV help er RNA provided all PV proteins and naturally occurring polyprotein precursors in trans Negative-strand synthesis was assayed as described above from reactions containing one of the template RNAs depicted in Figure 4-3A with an equimolar amount of PV1 GUA3 helper RNA. Interestingly, RNA templates which translated anything less than the complete P23 polyprotein exhibited a significant defect in their ability to su pport efficient negative-strand synthesis (Figure 4-3B, compare lanes 1-6 to lane 7). The replication deficient P23-3CSTOP RNA and the replication competent P23 RNA only diff er by the inclusion of the 3D coding region, implicating 3Dpol or a 3D-precursor as the cis protein requirement. Sin ce the majority of the 3D coding region was present in the replication de ficient P1-3D* RNA (Fi gure 4-2), it is highly unlikely that physical ribosome transit through this region is responsible for the observed effect. Taken together, these data str ongly indicate that efficient PV negative-strand synthesis requires cis translation of 3Dpol, 3CDpro, and/or another 3D containing precursor.

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91 Translation of the 2BCP3 Protein Precursor in cis is Sufficien t for Efficient PV Negative-strand Synthesis To determine if efficient PV negative-strand synthesis requires cis translation of 3Dpol alone or the translation of a larger 3D containing precursor, a second series of RNA templates was constructed. These constructs all contain the same 5 and 3 NTRs, however each RNA in the series contains the coding sequence for a succe ssively larger 3D contai ning precursor that has been previously associated with PV replica tion (Figure 4-4A) (104, 119, 159). In addition to these template RNAs, an additional control R NA was generated which contained an in-frame deletion of nucleotides 867-6011 from full-lengt h PV1 RNA [PV1p50 RNA]. This RNA retains the entire PV 5 and 3 NTRs, utilizes the au thentic start/stop codons and codon contexts, and translates a non-functional 50 kDa pr otein (p50), serving as an addi tional control for the effect of ribosome transit through the RNA. Here again, the full-length PV helper RNA provided all PV proteins and naturally occurri ng polyprotein precursors in trans Negative-strand synthesis was assayed as described above from reactions containing one of the template RNAs depicted in Figure 4-4A with an equimolar amount of PV1 GUA3 helper RNA. As shown in Figure 4-4B, RNAs which translated p50, 3Dpol, 3CDpro, 3BCD, or P3 proteins in cis were all unable to efficiently serve as RNA templates for negative-strand synthesis (Figure 4-4B, compare lanes 1-5 to lane 7). Strikingly, template RNA which translated the 2BC-P3 precursor protein in cis supported negative-strand synthesis to levels greater than that observed with P23 RNA (Figure 4-4B, compare lane 7 to lane 8). These data clearly demonstrate that neither cis translation of 3Dpol alone nor ribosomal transit through the 3D coding region of a template RNA is sufficient to promote efficient PV ne gative-strand synthesis on that template. The replication deficient P3 RNA and the replication competent 2BC-P3 RNA only differ by the inclusion of the 2BC coding regi on, which indicates strongly that efficient PV

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92 negative-strand synthesis requires cis translation of either the full 2BC-P3 precursor protein or a combination of individual, discrete protein functions within the 2BC-P3 polyprotein. If efficient PV negative-strand synthesis requires cis translation of multiple discrete proteins/precursors, one of the most likely candidate proteins (in addition to 3Dpol/3CDpro) is protein 2C. Protein 2C has been directly impli cated in negative-strand initiation and has been shown to specifically bind to PV RNA (17, 18, 122, 170, 202). Therefore, a 2C-P3 expressing template RNA was constructed and tested as was described above (Figure 4-5A), despite the fact that a 2C-P3 precursor protein has neither been ob served nor postulated to play a role in PV replication. Negative-strand synthesis was assayed as described above from reactions containing P3, 2C-P3, or 2BC-P3, as well as an equimolar amount of PV1 GUA3 helper RNA. As shown in Figure 4-5B, the 2C-P3 expressing template RNA supported nearly identical levels of negative-strand synthesis as the re plication deficient P3, and suppor ted significantly lower levels of negative-strand synthesis compared to those observed from the 2BC-P3 template RNA. Taken together, these results strongly implicate cis translation of the 2BC-P3 precursor as the primary requirement for efficient PV negative-stra nd synthesis. Multiple discrete proteins or protein precursors derived from 2B C-P3 which include the 2B and 3Dpol polypeptides may function synergistically to achieve efficient init iation of negative-strand synthesis. However, given the obligatorily sequential nature of the PV polyprotein, the 2BC-P3 precursor would be the minimum cis translation product require d in either case for efficient negative-strand RNA synthesis. Discussion The work presented here has clearly establis hed a close coupling of PV translation and initiation of negative-stra nd RNA synthesis in the in vitro HeLa S10 translation-replication

PAGE 93

93 system, mirroring the characte ristics of PV infection in vivo Moreover, we have demonstrated that this coupling is due to a marked cis preference of viral protein f unction, rather than the result of RNA template preparation induced by ribosomal transit. Through the use of trans complementation RNA replication assays in th e cell-free system, we have defined this cis acting protein product as the 2BC-P3 pr ecursor polyprotein. This result, in combination with previous observations by our lab and others, allows us to propose a model whereby the translation of the 2BC-P3 precursor in cis is followed rapidly by a concerte d association with newly formed membrane vesicles as well as its template RNA, initiating the critical process of replication complex assembly. This model provides mechanis tic insight into the f unctional coupling of PV translation and initiati on of RNA replication. PV Translation in cis is a Prerequisite for Efficient RNA Replication Using the HeLa S10 translation-replicati on reactions, we have observed that PV subgenom ic RNA which translates a ll its replication proteins in cis [P23 RNA] exhibits approximately 5 to 10-fold higher levels of negative-strand RNA synthesis than the similar frameshifted RNA [FS23 RNA], which obt ains its replication proteins in trans These observations are consistent with the previous finding that a ll naturally occurring defective interfering (DI) PV genomes maintain the transla tional reading frame, despite containing various deletions in the P1 coding region (115). Add itionally, previous work by our lab and others has shown that maintaining the read ing frame of PV RNAs through th e majority of the P23 coding sequence was required for efficient RNA replicati on in cell culture, even in the presence of a helper RNA or helper virus (61, 151, 197). This phenomenon has also been observed in cell culture during PV infection, in that genomes whose RNA replication has been arrested by guanidine will, following release of the guanidine block, return to the ER to re-start translation prior to RNA replication, despite the presence of suff icient PV proteins (70). In total, these data

PAGE 94

94 strongly indicate that PV RNA rep lication requires not only newly tr anslated replication proteins, but that these nascent proteins be translated in cis from the PV genome which will subsequently begin negative-strand RNA synthesis. It is possible that the observe d replication defect of PV R NAs resulting from prematurely aborted translation is a secondary effect of decreased RNA stabili ty. It is known that cellular mRNAs which are improperly translated are s ubject to a wide range of RNA degradative machinery, including nonsense mediated deca y (56, 146). Recent work by Kempf and Barton has also indicated that polyribosome assemb ly on PV RNA imparts some protection from endogenous exoribonucleases, sugges ting that the absence of polysomes would result in RNA degradation (107). Interestingl y, we observed no increase in th e degradation of RNAs which prematurely terminated translation. This obs ervation is again consis tent with previous observations by our lab and others that premat ure termination of PV RNA does not result in RNA instability (105, 151; Ogram et al ., unpublished results). These results show that the RNA replication defect observed in frameshifted PV RNAs is not a secondary effect of decreased RNA stability, but instead is a dire ct result of the incomplete cis translation of the replicating RNA. Complete Ribosome Transit Through a Templa te RNA is Not Sufficient to Promote High Levels of N egativestrand RNA Synthesis Because initiation of negative-strand RNA synt hesis and termination of translation both occur at the extreme 3 end of the PV genome, it is possible that complete ribosomal transit and translation termination in the authentic RNA contex t is required for prepar ing the 3 end of the genome for efficient initiation of negative-strand synthesis. Usi ng constructs which contain the authentic translation termination context but do not translate any functiona l replication proteins, we observed no significant diffe rence between RNA which transl ated its entire open reading

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95 frame (P1-3D*) compared to an RNA which term inated translation prematurely (FS1-3D*). This clearly showed that neither complete ribosome transit, nor authentic translation termination, conferred the ability to efficiently in itiate negative-stra nd RNA synthesis. A set of very elegant in vivo experiments was performed by Novak and Kirkegaard using PV RNAs containing premature amber stop codons in which replication was assayed in both non-permissive and amber-suppresso r cells (151). Using this system, Novak and Kirkegaard observed a severe replication defe ct in PV RNAs which prematur ely terminated translation and proposed a series of potential models to explain th eir results. One such model proposes that the act of ribosomal transit through a critical re gion of the template RNA promotes efficient negative-strand synthesis by affecting RNA s econdary structure or by affecting protein association with the RNA template. We observed that the cis translation of the P23-3CSTOP RNA or 2C-P3 RNA did not result in efficient initia tion of negative-strand sy nthesis, yet these two RNAs together contain the entire RNA sequen ce which comprises the critical region identified by Novak & Kirkegaard. This indicates that the efficient initiation of negative-strand RNA synthesis observed on fully translated template RNAs is a result of the cis action of a viral protein product(s) rather than the physical result of ribosomal tr ansit through a sp ecific region of the PV RNA. Poliovirus RNA Replication Re quires Translation of th e 2BC-P3 Pr ecursor in cis By providing all essential PV proteins and naturally occurring precursors in trans from a helper RNA, we assayed for negative-strand synthesis from PV subgenomic RNA expressing sequentially larger portions of the PV replicati on polyprotein. Using this additive approach, we determined that the protein product required in cis for efficient negative-strand synthesis was either 3Dpol or a 3D-containing precursor protein. By assaying for negative-strand synthesis from RNAs expressing increasingly larger 3D-containing precursor proteins, we determined that

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96 the 2BC-P3 precursor was the minimal PV polyprotein precursor required in cis to efficiently initiate negative-strand synthesis. This concept is consistent w ith previous work which showed that deletions extending past the 2Apro coding sequence are lethal in vivo and that providing 2Apro in trans is sufficient to fully complement a 2Apro deficient PV replicon RNA (61, 104, 105). Under conditions where 2Apro is being provided in trans such as those in Figure 4-4, the addition of the 2A polypeptide to the cis precursor (i.e. comparing 2 BC-P3 to P23) actually is disadvantageous, forcing an additional cleavage step prior to genera tion of the ideal precursor. This would explain the observati on that 2BC-P3 RNA replicates better than P23 RNA when in the presence of a helper RNA. It remains possible that, rather than the 2BC-P3 precursor in its entirety, it is the function of multiple distinct proteins or protein precursors within 2BC-P3 that are required in cis However, studies from the laboratory of Eckhard Wimmer showed that a dicistronic PV RNA containing the EMCV IRES between the 2A and 2B coding sequence generated viable virus and showed no abnormal polyprotein processing, whereas insertion of the EMCV IRES at any other intergenic position in the repli case polyprotein was lethal (139, 160). These studies clearly show that an intact 2BC-P3 precursor is essential for efficient PV RNA replication. Previous work from our lab further establishes the critical na ture of the 2BC-P3 precursor by showing that a lethal mutation in protein 2C (2C[P131N]) coul d only be complemented efficiently by 2BC-P3 and not by a smaller precursor (104). It is critical to not e, however, that despite the ability of the 2BC-P3 precursor to complement in trans the ability of 2BC-P3 to promote negative-strand RNA synthesis is still dramatically more efficient in cis than in trans. Taken together, these observations indicate that the entire 2BC-P3 precursor as the essential cis acting viral protein factor required for the efficient initia tion of negative-strand RNA synthesis.

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97 A Model for PV RNA Replication Complex Formation D ependent on cis Translation of the 2BC-P3 Precursor Polyprotein Poliovirus replication occurs on membra nous vesicles induced upon infection by a combination of hydrophobic viral proteins and prot ein precursors. Interestingly, when these vesicles are induced via heterol ogous expression of these proteins they are not utilized for RNA replication by a superinfecting virus (71). As th e authors of this previ ous study concluded, this indicates that membrane vesicles must be induc ed immediately prior to RNA replication, by the proteins produced from the genome about to be replicated. This would require at least cis translation of 2CATPase and/or 2BC, since it has been shown that characteristic membrane rearrangements are induced by thes e two proteins (9, 20, 50). It is also of note that the PV polyprotein is subject to two di stinct processing cascades as described by Lawson & Semler, one soluble pathway and one which is membrane a ssociated (119). The membrane associated processing pathway initiates with the creation of the 2BC-P3 precursor, which we have identified here as the critical cis acting PV protein responsible for efficient initiation of negative-strand RNA synthesis. Furthermore, gi ven the significantly short half -life of the 2BC-P3 precursor observed by Lawson & Semler, we assert that the trans acting capability of the 2BC-P3 precursor is severely restricted both by its inhe rently transient nature, as well as its membrane association. Based on our results presented abov e as well as work performed by multiple other laboratories, we propose a replic ation model whereby the PV polypr otein precursor 2BC-P3 acts in cis to bind its genomic RNA and simultaneous ly induce and associate with membrane vesicles, forming an active PV replication comple x. This concerted proc ess acts to functionally couple viral translation, membrane vesicle i nduction, and RNA replication, and represents a critical transition in the PV life-cycle from genomic translation to RNA replication.

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98 Figure 4-1. Translation of a PV RNA template is a prerequisite for efficient negative-strand synthesis. A) Schematic of poliovirus RNAs used in these experiments. PV1 GUA3 helper RNA contains the entire PV RNA sequence with a mutation in the 3'NTR, rendering it incapable of RNA replicati on. P23 RNA encodes all essential viral replication proteins, and FS 23 RNA contains a frameshift mutation in the 2A coding region of P23 RNA and does not express any functional protein B) Replication of P23 RNA and FS23 RNA was measured us ing PIRCs isolated from HeLa S10 reactions and RNA product was analyzed by CH3HgOH gel electrophoresis and autoradiography as described in Chapter 2. Each transcript RNA contained two non-viral 5 G residues which permits onl y negative-strand RNA synthesis. Equimolar amounts of PV1 GUA3 RNA were included in each reaction to provide all naturally occurring viral proteins in trans C) The stability of uniformly radiolabeled P23 RNA or FS23 RNA in HeLa S10 reactions was measured as described in Chapter 2. Aliquots of the reaction mi xtures were removed after the indicated incubation time and the full-length RNA remaining was analyzed by denaturing CH3HgOH gel electrophoresis and autoradiography. As before, an equimolar amount of PV1 GUA3 RNA was included in each reaction to r ecapitulate replication reaction conditions. Gels depicted in panels B & C we re generated by Dr. Nidhi Sharma.

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99 Figure 4-2. Physical ribosome tr ansit of a template RNA is not sufficient to promote efficient initiation of negative-strand synthesis. A) Schematic of poliovirus RNAs used in this experiment. P1-3D* RNA encodes a fusi on protein between the P1 coding region and a non-functional carboxy-terminal portion of 3D. During translation of P1-3D* ribosomes completely transit the length of the template and terminate translation in the authentic RNA context. FS1-3D* RNA contains a frameshift mutation early in the P1 coding region of P1-3D* RNA and terminates translation prematurely, without completely transiting the RNA template. B) Replication of P1-3D* and FS1-3D were assayed in the presence of equimolar amounts of PV1 GUA3 helper RNA using PIRCs isolated from HeLa S10 reactions as described in Chapter 2. RNA product was visualized by denaturing CH3HgOH gel electrophoresis and autoradiography. The gel depicted in panel B wa s generated by Dr. Nidhi Sharma.

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100 Figure 4-3. Translation of 3D or a 3D precursor is required in cis for efficient initiation of negative-strand synthe sis. A) Schematic of poliovirus RNAs used in this study. Each successive templa te RNA encodes one additional protein component of the PV replication polyprotein (P23), such that P23-2ASTOP encodes only 2A, P23-2BSTOP encodes 2AB, P23-2CSTOP encodes 2ABC, and so on. All RNAs used in these experiments are id entical in length, and differ in sequence only by the inclusion of two stop codons at the indicated positi on in the coding region. B) Replication of each template RNA indicated above was assayed in the presence of equimolar amounts of PV1 GUA3 helper RNA as described previously. Full length product RNA was analyzed by denaturing CH3HgOH gel electrophoresis and levels of negative-strand synthesis were quantitated by phosphorimager. The level of negative-strand synthesis of each RNA we re scaled relative to those observed with P23 RNA and represented graphically below the autora diograph. The gel depicted in panel B was produced by Dr. Sushma Ogram.

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101 Figure 4-4. Efficient initiation of ne gative-strand synthesis requires transla tion of 2B or a 2B precursor in cis A) Schematic of poliovirus RNAs used in this study. Each RNA encodes a successively larger 3Dpol precursor as indicated by the template name listed at left. PV1p50 RNA acts as a control RNA, translating a non-functional 50 kDa protein and utilizing the authentic translational start and stop cont exts. B) Replication of each template RNA indicated above was assayed in the presence of equimolar amounts of PV1 GUA3 helper RNA as described previ ously. Full length product RNA was analyzed by denaturing CH3HgOH gel electrophoresis and quantitated by phosphorimager. Th e levels of negative-strand synthesis of each RNA were scaled relative to P23 RNA and are represented graphically below the autoradiograph.

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102 Figure 4-5. Poliovirus RNA replic ation requires translation of th e 2BC-P3 polyprotein precursor in cis A) Schematic of the poliovirus RNAs used in this study. P3 RNA encodes the P3 polyprotein precursor, 2C-P3 RNA en codes the 2C-P3 precursor, and 2BC-P3 RNA encodes the 2BC-P3 precursor. B) Re plication of each template RNA indicated above was assayed in the presen ce of equimolar amounts of PV1 GUA3 helper RNA as described previously. Full length product RNA was analyzed by denaturing CH3HgOH gel electrophoresis and quantitat ed by phosphorimager. The levels of negative-strand synthesis of each RNA were scaled relative to 2BC-P3 RNA and are represented graphically be low the autoradiograph.

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103 CHAPTER 5 MUTLIPLE MOLECULES OF THE 3CD VIRAL PROTEIN PRECURSOR PERFORM DISCRETE FUNCTIONS IN THE I NITIATI ON OF POLIOVIRUS NEGATIVE-STRAND RNA SYNTHESIS Introduction The differential use of polyprotein precursors an d their products is a key strategy em ployed by poliovirus (PV) to perform th e many diverse functions required during viral rep lication using limited sequence space. An extension of this is the evolution of multip le activities within a single protein or protein precu rsor. The PV precursor 3CDpro exemplifies both of these concepts in that it performs multiple functions as a precur sor and these activities are functionally distinct from is processed products 3Cpro and 3Dpro. As a precursor, 3CDpro exhibits no polymerase acitiv ity, however its processed product 3Dpol, acts as the R NA-d ependent R NA p olymerase (RdRp) (73, 74, 88). The 3CDpro precursor also has the ability to bind to stem-loop d of the 5CL, and while this ability is partially retained by its processed product 3Cpro, the binding affinity of 3Cpro for the 5CL is 10-fold lower than that of 3CDpro (12). And while both 3CDpro and 3Cpro are proteases, their cleavage specificities and activity levels are different, a nd this difference is particularly apparent in the processing of the viral capsid precursor (P1) and at the 3C-3D junction (157). In these cases, 3Cpro processing of P1 and 3CD were 1000-fold and 100-fold le ss efficient than the processing observed by 3CDpro. Interestingly, there are very fe w structural differences between 3Cpro and 3Dpol alone and within the 3CDpro precursor as determined by x-ray crystallography.(130). The current model of PV repl ication complex formation i nvokes genomic circularization mediated by RNP complexes formed at the 5CL and 3NTR/poly(A) tail to promote initiation of negative-strand synthesis (26, 93, 126, 196). Given that 3CDpro, in the presence of PCBP and/or 3AB, was observed to form RNP complexes with the 5CL as well as the 3NTR (12, 14, 89,

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104 158, 213), these circular models also included two molecules of 3CDpro. It has been established that the formation of the 5CL-3CDpro RNP complex is required fo r negative-strand synthesis (12, 158, 213), but the func tional role of 3CDpro bound to the 3NTR is yet to be elucidated. The consequences of differentia l interactions between 3CDpro and either 3AB or PCBP have also not yet been addressed. Additionally, it has not yet been determined if the molecule binding to the 5CL is the same as that which binds the 3NTR or if these are indeed two different molecules as has been modeled. Although it has been established that th e above described activities of 3CDpro are required for PV RNA replication, the precise molecular mechanisms which drive these requirements have not been delineated (12, 89, 158, 213). In an effort to more directly characterize some of these mechanisms, we utilized the HeLa S10 tran slation-replication system to examine the complementation profiles of functionally define d mutants in the PV protein precursor 3CDpro. To do this, we performed trans complementation analysis of PV subgenomic RNA replicons containing lesions in the 3CDpro coding sequence. These mutant RNAs were assayed for their ability to assemble functional replication comple xes and initiate negative-s trand synthesis in the presence of complementing protein expressi on RNAs. Herein, we demonstrate that 3Dpol must be admitted into the replication comp lex as its immediate precursor 3CDpro and that binding to the 5CL is not required for th is activity. In addition, we provide compelling evidence that at least two molecules of 3CDpro are present in the PV replicat ion complex and these individual precursors perform multiple distin ct functions. Lastly, we show that the 5CL RNP complex essential for initiation of negative-strand synthe sis is likely formed by a molecule of 3CDpro which enters the replication complex in the form of its precursor P3.

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105 Results Mutations Which Prevent the Production of Active 3Dpol are Rescued by 3CDpro To begin to analyze the role of 3CDpro in the formation of the PV replication complex used to initiate negative-strand RNA synthesis, we first needed to establish the phenotypes of each specific 3CDpro mutants in the HeLa S10 translation-re plication system. The first subtype of 3CDpro mutants examined were those which failed to generate active 3Dpol. The first of these was a previously described mutant in the highly conserved YGDD mo tif of RNA-dependent RNA polymerases, where the Gly327 of PV 3Dpol was mutated to Met (3D[G327M]) (98). This mutation was shown to abolish a ll polymerase activity in bacterially expressed recombinant PV 3Dpol. The second mutant 3CDpro examined contained four sequential mutations of the 3C-3D cleavage site, all on the 3Cpro side of the junction in positi ons P1-P4, thereby maintaining the integrity of the 3Dpol amino acid sequence. This proces sing mutant (3CD[PM]) combines two previously described processing site mutations [T181K, Q182D] with two additional mutations [S183G, Q184N] designed to completely abroga te 3C-3D processing (12, 37, 88). This extensive mutagenesis is required to completely inhibit processing of 3CDpro, as individual mutations as well as combinations thereof ha ve been shown to reduce, but not eliminate processing ((88); data not shown). Because the 3CDpro precursor does not possess any of the polymerase activity of its progeny 3Dpol, the abrogation of 3CD pr ocessing also functionally inactivates polymerase activity (73, 74, 88). Since both 3CD[G327M] an d 3CD[PM] would be unable to generate a functional 3Dpol, PV RNAs containing these mu tations should be unable to replicate. Each mutant 3CDpro was assayed for its ability to support negative-strand RNA synthesis of a previously described subgenomic PV RNA replicon (P23 RNA) which contained the above described mutations in the 3CD coding region. Negative-strand synthe sis was assayed using

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106 PIRCs isolated from HeLa S10 translation-replication reactions as de scribed in Chapter 2. Radiolabeled full-length product R NA was visualized by denaturing CH3HgOH gel electrophoresis and autoradiography as previous ly described. As expected, P23 RNA which expresses wild-type 3CDpro generated significant amounts of negative-strand product RNA (Figure 5-1A, lane 1). However, P23 RNAs which express a 3CDpro that cannot generate active 3Dpol are unable to generate detectible levels of negative-strand s ynthesis (Figure 5-1A, lanes 2-3). To ensure that the observed RNA replication phenotype of these mutants was not the result of defects in translation or pro cessing, protein synthesis in the replication reactions was analyzed by [35S]methionine incorporation, SDS-PAGE, and autoradiography. As shown in Figure 5-1B, both mutant P23 RNAs generate similar levels of protein synthesis. Further, there are no significant differences in the pattern of polyprotein processing, except where 3Cpro and 3Dpol were absent from reactions expressing the 3CD[PM ], as expected (Figure 5-1B, lane 3). These data confirm that mutations which prevent the generation of active 3Dpol block PV negative-strand RNA synthesi s, and the mutations tested do not affect translation or polyprotein processing. To determine if these 3Dpol deficient mutations can be complemented in trans negative-strand synthesis of P 23-3D[G327M] RNA or P23-3CD[ PM] RNA was assayed in the presence of non-replicating helper RNAs encoding sequentially larger 3D containing precursors. Levels of negative-strand synthesis were assessed as described above. Interestingly, expression of wild-type 3Dpol alone was not sufficient to rescue negative-strand synthesis to significant levels for either of the 3Dpol deficient mutants (Figure 52A/2C, lane 1). However,

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107 negative-strand synthesis of these mutants was efficiently restored by complementation with 3CDpro or a larger precursor (F igure 5-2A/2C, lane 2-4). Protein synthesis and polyprotei n processing in the replication reactions was also analyzed by [35S]methionine incorporation, SD S-PAGE, and autoradiography. Despite the presence of a disproportionate excess of polymerase in reactions which ex pressed solely 3Dpol (Figure 5B/5D, lane 1), only minimal levels of negative-strand synthesis were observed in complementation assays. Furthermore, there is direct correlation between the amounts of 3CDpro present and the level of trans complementation observed in RNA replica tion assays. Taken together, these data suggest that the active 3Dpol is delivered to the replication complex in the form of its immediate precursor, 3CDpro. Complementation of 3Dpol Deficient Mutations Requires the Intact 3CDpro Precursor To further characterize the complementation of the 3Dpol deficient mutations by 3CDpro, negative-strand synthesis of P23 RNAs expr essing either 3CD[G327M] or 3CD[PM] was assayed in the presence of a combination of 3Cpro and 3Dpol expression RNAs or an RNA which expresses the heterologous mutant 3D/3CD. Levels of negative-strand synthesis were assessed as described above. As before, complementation of both 3Dpol deficient mutants by 3CDpro was significantly more efficient than complementation by 3Dpol (Figure 5-3A/3C, lanes 1-2). Complementation using a combination of 3Cpro and 3Dpol expression RNAs was slightly less efficient than using a 3Dpol expression RNA alone, and negative-strand synthesis was undetectable in complementation reactions containing the heterologous 3D/3CD mutant expression RNA (Figure 5-3A/3C, lanes 3-4). Analysis of prot ein synthesis and polyprotein processing showed equal levels of protein synt hesis and processing, except where expected for additional proteins expressed in trans (Figure 5-3B/3D). These results clearly show that the

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108 active 3Dpol used to initiate negative-strand RNA synt hesis is delivered to the PV replication complex in the form of its intact 3CDpro precursor. Mutations Which Disrupt 3Cpro/3CDpro Binding to the 5CL Block RNA Replication and Affect Polyprotein Processing To further analyze the role of 3CDpro in the formation of the PV replication complex used to initiate negative-strand RNA synthesis, we generated mutants in the RNA binding region of 3Cpro/3CDpro and determined the replication and transl ation phenotypes of these mutants in the HeLa S10 translation-replicati on system. Three distinct regi ons of the 3C primary sequence have been implicated in binding to the 5CL, an N-terminal region (Y6, K12, R13), a central region (K82, F83, R84, D85, I86, R87), and a Cterminal region (T154, G155, K156) (12, 33, 36, 132, 142). The residues included in the C-te rminal RNA binding region have also been implicated VPg uridylylation on the cre(2C) hairpin and overlap a predicted protein-protein interaction site (130), making th is region unattractive for mutagenesis. The residues in the central region represent a highly conserved pi cornaviral KFRDIR 3C-RNA binding motif, and a previously described mutant, 3C[R84S], has been included in this analysis as a prototypic example of mutations in this re gion(36). Lastly, a double mutant in the N-terminal RNA binding region of 3Cpro was also created which combined tw o adjacent previously described RNA binding mutations, 3C[K12N/R13N] (36). Each 3CD R NA b inding m utant (RBM) was assayed for its ability to support negative-strand RNA synthesis of P23 RNA which contained one the above described mutations in the 3CD coding region. The levels of nega tive-strand synthesis from isolated PIRCs were assessed as described above. As shown previ ously, P23 RNA which expresses wild-type 3CDpro generated significant amounts of negative-strand pr oduct RNA (Figure 5-4A, lane 1). However,

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109 P23 RNAs which express a 3Cpro/3CDpro that cannot bind the 5CL are unable to generate detectible levels of negative-stra nd synthesis (Figure 5-4A, lanes 2-3). To determine the translational and proce ssing phenotypes of these mutants, protein synthesis in the replication reactions was analyzed by [35S]methionine incorporation, SDS-PAGE, and autoradiography. As shown in Figure 5-4B, both mutant P23 RNAs generate similar levels of protein synthesis, however, bot h mutants exhibited differences in the pattern of polyprotein processing (Figure 5-1B lane 2-3). In reactions containing P23-3C[R84S], there was a significant accumulation of unprocessed high molecular weight precursors. Likewise, every mutation tested within the conserved KFRD IR motif exhibited some degree of polyprotein processing defect (data not shown), which comp licates the interpretation of RNA replication phenotypes. In reactions contai ning P23-3C[K12N/R13N], there was a moderate but detectable increase in the efficiency of 3C-3D processing, however this is likely benign, particularly since all other polyprotein processing seems unaffecte d. These data, particularly the 3C[K12N/R13N] mutant, indicate that mutations which disrupt the binding of 3Cpro/3CDpro to the 5CL block PV negative-strand RNA synthesis Complementation of 3Cpro/3CDpro RNA Binding Mutants Re quires the Intact 3CDpro Precursor Although 3Cpro can bind RNA, it has been shown that the 3CDpro precursor has a 10-fold higher affinity for the PV 5CL than does 3Cpro alone (12). And given the significant excess of 3CDpro over 3Cpro that exists during PV infection, the most likely 5CL RNP complex is one which contains 3CDpro rather than 3Cpro alone. To test this assu mption, complementation assays were performed using P23 RNA containing either the 3C[K12N/R13N] or 3C[R84S] mutations, in combination with non-replicating helper RNAs expressing either 3Cpro alone or its precursor

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110 3CDpro. In addition, the mutant P23 RNAs were each complemented with a combination of 3Cpro and 3Dpol expressing RNAs or an RNA whic h expressed the heterologus 3CDpro[RBM]. As predicted, the defect in negative-strand synthesis of both P23[RBM] RNAs were unable to be complemented by 3Cpro alone or 3Cpro and 3Dpol in combination (Figure 5-5A, lanes 1, 3, 5, and 7). In contrast, expression of the 3CDpro precursor was able to complement both P23-3C[K12N/R13N] as well as P23-3C[R84S], while the he terologous 3CD[RBM] was unable to do so (Figure 5-5A, lanes 2, 4, 6, and 8). It is of note, however, that the efficiency of rescue differed significantly between the two mutants, most likely as a result of interference by the defective polyprotein proce ssing exhibited by 3CD[R84S]. Analysis of protein synthesis in these r eactions shows appropria te expression of all complementing proteins as well as similar levels of protein synthesis fo r both P23[RBM] RNAs (Figure 5-5B/5C). Interestingly, some of the polyprotein processing defect exhibited by P23-3C[R84S] is complemented in trans by 3Cpro/3CDpro, and this complementation is even greater in the reaction which expressed 3CD[K12N/R13N] which exhibited elevated processing activity (Figure 5-5C). Despite these minor processing irregulariti es, these results clearly show that the intact 3CDpro precursor is require d to complement a 3Cpro RNA binding mutation. Moreover, this confirms that 3CDpro, and not 3Cpro, is a component of the 5CL RNP complex required for the initiation of negative-strand synthesis. Complementation Between Two Functionally Distinct 3CDpro Mutants Work by Cornell et al. showed reciprocal complementa tion between an RNA expressing a non-functional chimeric polymerase and an RNA expressing a P3 precurs or which contained a 3C/3CD RNA binding mutation (62). The author s therefore concluded that viral proteins capable of binding RNA and initia ting replication complex formation, can recruit complementing proteins to the replication via protein-protein interactions. In the context of the current

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111 investigation, this would predict that a 3Dpol deficient 3CDpro mutant which retained RNA binding ability could be complemented by a 3CD[RBM] which could generate functional polymerase. Replication complexes would ther efore require the presence of at least two molecules of 3CDpro for such a complementation to occur, and the potential replication complex models for this are depicted in Figure 5-6. To determine if reciprocal complement ation of two functionally distinct 3CDpro mutants was possible, negative-strand RNA synthesi s was assayed in reactions containing P23-3CD[G327M] RNA in combination with a he lper RNA which expressed a wild-type or mutant 3CDpro. For the purposes of these complementation experiments, 3CD[K12N/R13N] was the RNA binding mutant of choice du e to the severe processing de fects exhibited by 3CD[R84S]. In reactions containing P23-3D [G327M], efficient complement ation was observed in the presence of wild-typ e or K12N/R13N 3CDpro RNA, but not in the presence of the synonymous 3CD[G327M] RNA (Figure 5-7A lanes 1-3). When the complementation was reversed, P23-3CD[K12N/R13N] was able to be comp lemented by both wild-type and G327M 3CDpro RNA, but not by the synonymous 3CD[K12N/R13N] RNA (Figure 5-7A, lanes 4-6). However, the reversed complementation efficiency was si gnificantly reduced relative to the original complementation, even in th e presence of wild-type 3CDpro helper RNA. To determine if the above complementati on would also occur for the similarly 3Dpol deficient 3CD[PM], negative-strand RNA synt hesis was assayed in reactions containing P23-3CD[PM] RNA in combination with a helper RNA which expressed a wild-type or mutant 3CDpro. As expected, efficient complementa tion was observed in reactions containing P23-3D[G327M] RNA in the presence of wild-type or K12N/R13N 3CDpro RNA, but not in the presence of the synonymous 3CD[PM] RNA (Figure 5-7C, lanes 1-3). And here too, when the

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112 complementation was reversed, the same pattern a nd decrease in efficiency of complementation was observed as was for the 3CD[G327M ] mutation (Figure 5-7C, lanes 4-6). To determine if the differences in complementation efficiencies were caused by abnormalities in translation and/or polyprotein processing, protein synthesis was monitored by [35S]methionine incorporation and assessed by SD S-PAGE and autoradiography as before. As shown in Figures 5-7B and 5-7D, overall protein synthesis was nearly identical in all reactions and polyprotein processing showed no abnormalit ies (except for expected differences for 3CD[K12N/R13N] and 3CD[PM] as previously discussed). These results clearly show that two distinct mutations in essential functions of the 3CDpro precursor can be reciprocally complemented to restore replication complex formation and negative-strand synthesis. Therefore, two or more molecules of the 3CDpro precursor must be simultaneously present in the PV replication complex, such as diagrammed in Figure 5-6D. High Efficiency Complementation of 3C [K12 N/R13N] Requires the P3 Precursor Interestingly, although the same pattern of complementation was pr esent, we observed a significant decrease in complementation effici ency when the template RNA contained the 3CD[K12N/R13N] mutations. This could result from either a dominant negative effect of a larger 3CD[K12N/R13N] containi ng precursor, a more stringent requirement for proteins in cis to bind the 5CL, or the requirement of a larger precursor to provide RNA binding in trans To test this, we first assessed the ability of P23-3C[K12N/R13N] RNA to be complemented by RNAs expressing sequentially larger 3Cpro containing precursors. As before, in reactions containing P23-3C[K12 N/R13N] RNA and 3Cpro expression RNA, levels of negative-strand synthesis were undetectable, whereas complement ation was observed in the presence of RNA which expressed 3CDpro (Figure 5-8A, lanes 1-2). Surprisingly, higher levels of negative-strand synthesis were observed when P23-3C[K12N/R13N] RNA was complemented with P3 or

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113 2BC-P3 expression RNA (Figure 5-8A, lanes 3-4) Aside from the expected increases in the levels of additional viral proteins expressed in trans there were no signifi cant alterations in the translation or polyprotein processi ng profile in any of the replication reacti ons (Figure 5-8B). These results clearly show that high efficiency complementation of a 3CDpro RNA binding mutant requires the presence of a P3 precursor Moreover, these data suggest that the P3 precursor delivers 3CDpro to the 5CL during the formation of the 5CL RNP complex which is essential for the initiation of negative-strand RNA synthesis. Discussion The work presented h ere has clearly illust rated the multifunctional nature of the viral 3CDpro precursor, particularly as it pertains to the initiation of negative-strand RNA synthesis. By performing trans complementation assays using the HeLa S10 translation-replication system, we have further defined the role of 3CDpro, as well as its precursors and processed products, in the formation of a functional PV replication co mplex. Using this approach, we have clearly shown that 3Dpol is admitted to the replication complex in the form of its intact immediate precursor 3CDpro and that binding to the 5CL is not a pr erequisite for this activity. Furthermore, by performing reciprocal complementation using two 3CDpro mutants in distinct, essential functions, we have shown that there are at least two molecules of 3CDpro present in the PV replication complex which perfor m discrete functions. Lastly, we have shown that the 3CDpro which forms the essential 5CL RNP complex is lik ely admitted to the replication complex in the form of its precursor P3. Active 3Dpol is Admitted to the PV Replication Complex in the Form of its Polymerase-inactive Precursor 3CDpro Although 3CDpro contains the entire 3Dpol peptide, it contains none of the associated polymerase activity (73, 74, 88). This is most likely due to changes in positioning of the

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114 N-terminus of 3Dpol that occur subsequent to pro cessing (95, 130, 180, 199). This strategy allows PV to synthesize large amounts of 3CDpro prior to replication without risking the generation of non-specifc dsRNA products on ce llular mRNAs which could activate innate immune pathways. Here, the 3CDpro precursor functions as a pro-enzyme which can be synthesized to high-levels and act ivated rapidly on demand. We we re able to show that mutants in either 3C-3D processing or the conserved 3Dpol RdRp motif could only be rescued efficiently by an intact 3CDpro or a 3CD containing precursor This indicates that 3CDpro is recruited into the PV replication complex in an inactive form and its activation by processing represents the firing of replication comple xes and marks the initiation of negative-strand synthesis. Additionally, this recruitment does not require direct binding of the 3CDpro precursor to RNA, since mutations which disrupted conserved RNA binding residues in 3C were able to complement 3Dpol deficient RNA replicons. RNA Binding and Protease Activities of 3CDpro are Functionally Linked Each RNA binding mutation tested, in addition to its replication phenotype, also exhibited altered patterns of polyprotein processing. In most cases, this alteration was detrimental and resulted in accumulation of unprocessed precurs ors, however in one case (3C[K12N/R13N]) the mutations resulted in increased processing efficiency. The effect of the latter mutations was mild and manifested primarily as an increased proportion of processed 3Cpro and 3Dpol in replication reactions. These observations are consistent with recent structural work by Claridge et al. who showed that RNA binding by rhinovirus 3Cpro induced conformational changes in regions involved in proteolysis (53). In this manner, one face of 3Cpro/3CDpro communicates with the other to transmit information regarding RNA binding status to the proteolytic machinery. This has significant implications for the PV life-cycle, since the rapi d polyprotein processing that is observed in the membrane associated proces sing cascade may actually be performed by the

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115 5CL-3CDpro RNP complex. Since this processing path way is associated with RNA replication, polyprotein processing, membrane association, and replication comple x formation may be additionally coupled by enhanced proteolysis by RNA-associated 3Cpro/3CDpro. By this model, most RNA binding mutations which disrupt processing may essentially lock 3Cpro/3CDpro in an unbound conformation, whereas the K 12N/R13N mutant induces c onformational shifts that simulate the bound conformation in the absence of RNA. Multiple 3CDpro Peptides are Present in the PV RNA Replication Complex Used to Initiate Negative-strand RNA Synthesis Current models of initiation of PV negativestrand RNA synthesis i nvolve interaction of the 5 and 3 ends of genomic RNA, mediated by RNP complexes, to form a circular replication complex (26, 93, 126, 196). It was known that 3CDpro, in the presence of PCBP and/or 3AB, could form RNP complexes with both the 5 CL and the 3NTR (12, 14, 89, 158, 213). Based on this, in combination with our own 3D complement ation data, our model fo r circular replication complex formation included two molecules of 3CDpro. Later studies by Cornell et al. showed that negative-strand synthesis of an RNA encoding an inactive chimeric 3Dpol could be complemented by expressing a P3 precursor with an RNA binding mutation (62). However, the authors did not characterize in which function the chimeric polymerase was defective and examined only the P3 precursor fo r its ability to complement in trans From this data it is difficult to draw precise conclusions about re plication complex formation and composition. Using precise mutations which inacti vated single functions of the 3CDpro precursor, we demonstrated reciprocal complementation of 3Dpol deficient mutants (3CD[G327M] and 3CD[PM]) with an RNA binding mutant (3CD[K 12N/R13N]). Each of the mutants blocks negative-strand RNA synthesis as each represents a mutation(s) in a discrete but essential function of the PV replication complex. Since bo th functions are required simultaneously in the

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116 initiation of negative-strand synthesis, complement ation of these mutants requires that at least one copy of each mutant 3CDpro be present at the time of repli cation initiation. This represents the first conclusive functional evidence that multiple molecules of the 3CDpro polypeptide are present and perform discrete functi ons within the PV replication comp lex that is used to initiate negative-strand RNA synthesis. The 3CDpro Bound to the 5CL is Admitted to the PV Replication Complex in the Form of its Precursor P3 We observed that, although functional, RNAs expressing 3CDpro, 3CD[G327M], or 3CD[PM] were only capable of minimally comp lementing P23-3CD[K12N/R13N] RNA. This was significant because when the complement ation had been reversed, 3CD[K12N/R13N] expression RNA was capable of complementing P23-3D[G327M] and P23-3CD[PM] RNAs to significantly higher levels. Upon ex amining the ability of larger 3Cpro precursors to rescue negative-strand synthesis of P23-3C[K12N/R13N ] RNA, we showed that complementation efficiency was significantly higher in the presen ce of either P3 or 2BC-P3 expression RNAs. Given that expression of P3 resulted in the highe st level of negative-strand synthesis, and that expression of 2BC-P3 also provide s P3, we conclude that the 3CDpro which forms the essential RNP complex with the 5CL is first admitted into the replication complex in the form of the P3 precursor. This is particul arly interesting, since 3CDpro has been shown to bind to the 5CL in the presence of the 3AB precursor (89, 213). Together, 3AB and 3CDpro comprise the P3 precursor, which may enter the replication co mplex intact and subsequently process upon binding to the 5CL. Furthermore, since VPg(3B) serves as protein primer for RNA synthesis, the 3AB generated from above described P3 proc essing, could serve as the precursor for the VPg used to prime negative-strand synthesis. This model is consistent with previous work which

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117 showed that mutations in VPg wh ich blocked its priming ability could only be complemented in trans by P3 or in cis as an intragenic fusion to a 3CDprocontaining precursor (124).

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118 Figure 5-1. Mutations which prev ent the generation of active 3Dpol block RNA replication. A) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10 reactions as described in Chapter 2. Reactions contained subgenomic P23 RNA containing either a wild-type or mu tant 3CD coding region (3D[G327M] or 3CD[PM]). Full length RNA produ ct was analyzed by denaturing CH3HgOH gel electrophoresis and autoradiography. B) A portion of the HeLa S10 reactions described in (A) was metabolically labeled with [35S]methionine to assay for protein synthesis. These reactions were analy zed by SDS-PAGE and autoradiography. Viral proteins are indicated at left.

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119 Figure 5-2. Viral Precursor 3CDpro complements both 3D[G327M] and 3CD[PM] in trans A & C) Negative-strand synthesis was assaye d using PIRCs isolated from HeLa S10 reactions as described in Chapter 2. Reactions contai ned P23 RNA with a mutated 3CD coding region (3D[G327M] or 3CD[PM ]) and a second complementing RNA expressing the indicated protein. All complementing RNAs contain the GUA3 mutation which inhibits ne gative-strand synthesis. Product RNA was analyzed by denaturing CH3HgOH gel electrophoresis and auto radiography. B & D) A portion of the reactions described above was metabolically labeled with [35S]methionine and these reactions were analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left.

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120 Figure 5-3. Complementation of 3D[G327M] or 3CD[PM] requires the intact 3CDpro precursor. A & C) Negative-strand synthesis was assaye d using PIRCs isolated from HeLa S10 reactions as described in Chapter 2. Reactions contai ned P23 RNA with a mutated 3CD coding region (3D[G327M] or 3CD[PM ]) and a second complementing RNA expressing the indicated pr otein(s). All compleme nting RNAs contain the GUA3 mutation which inhibits ne gative-strand synthesis. Product RNA was analyzed by denaturing CH3HgOH gel electrophoresis and auto radiography. B & D) A portion of the reactions described above was metabolically labeled with [35S]methionine and these reactions were analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left.

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121 Figure 5-4. Mutations which disrupt 3Cpro/3CDpro binding to the 5CL block RNA replication. A) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10 reactions as described in Chapter 2. Reactions contained subgenomic P23 RNA containing either a wild-type or mutant 3C coding region (3C[K12N/R13N] or 3C[R84S]). RNA product wa s analyzed by denaturing CH3HgOH gel electrophoresis and autoradiography. B) A portion of thes e reactions described was metabolically labeled with [35S]methionine to assay for protein synthesis. These reactions were analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left.

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122 Figure 5-5. Complementation of 3C[K12N/R13N ] or 3C[R84S] requires the intact 3CDpro precursor. A) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10 reactions as described in Chap ter 2. Reactions contained subgenomic P23 RNA containing a mutant 3C coding region (3C[K12N/R13N] or 3C[R84S]) and a second complementing RNA expressing the indicated protein(s). RNA product was analyzed by CH3HgOH gel electrophoresis and au toradiography. B-C) A portion of the above reactions was metabolically labeled with [35S]methionine to assay for protein synthesis. These reactions were analyzed by SDS-PAGE and autoradiography. Viral protei ns are indicated at left.

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123 Figure 5-6. Schematic of trans complementation using two functionally distinct mutations in 3CDpro. A) In the presence of only 3CD[G327 M], replication complexes could form and process, however, the 3Dpol generated is catalytically inactive and RNA replication is blocked. B) In the presence of p rocessing m utant 3CDpro (3CD[PM]), replication complexes could form, however, 3CD has no polymerase activity before it is processed. Since 3CD[PM] cannot proce ss, negative-strand synthesis is blocked. C) In the presence of R NA b inding m utant 3CDpro (3CD[RBM]), the essential RNP complex at the 5CL cannot be formed, and as a result, negative-strand synthesis is blocked. D) If a 3CD[RBM] is co-expre ssed with either 3CD[G327M] or 3CD[PM], the polymerase deficient precursor could bind the 5CL and the RBM could provide the polymerase. This would allow in itiation of negative-strand synthesis.

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124 Figure 5-7. Two functionally distinct 3CDpro mutants can complement each other in trans A & C) Negative-strand synthesis was assaye d using PIRCs isolated from HeLa S10 reactions as described in Chapter 2. Reactions contai ned P23 RNA with a mutated 3CD coding region (3D[G327M]/3CD[PM ] or 3C[K12NR13N]) and a second complementing RNA expressing the indica ted protein. All complementing RNAs contain the GUA3 mutation which inhibits negativ e-strand synthesis. Product RNA was analyzed by CH3HgOH gel electrophoresis and autoradiography. B & D) A portion of the reactions described above was labeled with [35S]methionine and analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left.

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125 Figure 5-8. Complementation of a 3CDpro RNA binding mutant is more efficient when P3 is provided in trans A) Negative-strand synthesis was assayed using PIRCs isolated from HeLa S10 reactions as describe d in Chapter 2. Reactions contained P23[K12N/R13N] RNA and a second complementing RNA expressing 3C or 3C precursors of increasing size. All complementing RNAs contain the GUA3 mutation which inhibits ne gative-strand synthesis. Product RNA was analyzed by denaturing CH3HgOH gel electrophoresis and auto radiography. B) A portion of the reactions described abov e was labeled with [35S]methionine and these reactions were analyzed by SDS-PAGE and autoradiography. Viral proteins are indicated at left

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126 CHAPTER 6 SUMMARY AND CONCLUSIONS In this dissertation, I have presented and di scussed the results from three distinct yet interconnected lines of investigation into the pr otein requirem ents for the initiation of poliovirus negative-strand RNA synthesis. Each of these st udies has generated signif icant insight into how these key viral and cellular proteins function in PV replication complex assembly, and also to the broader understanding of the replication of rela ted enteroviruses. Techniques developed to perform this work have already been applied to the study of other stages of the viral life cycle, including PV translation and cre(2C)-dependent VPg uridylylati on, and will soon be adapted for characterization of Coxsackievirus B3 replication. Future work ba sed on each of these lines of investigation will provide a more detailed unders tanding of the molecular mechanisms by which poliovirus, as well as other enteroviruses, regula te the critical steps of viral RNA replication. The Role of PCBP in the Initiation of Poliovirus Negative-strand Synthesis The first of the investigations presente d herein probes the involvem ent of the multifunctional cellular protein PCBP in virus replication. To do so, our laboratory has developed and applied a novel prot ein-RNA tethering system to study of virus replication. Using this system we were able to confirm the activ ity of PCBP in supporting negative-strand synthesis and were further able to identify the functional domains within PCBP2. Morover, we were able to show that some, but not all members of th e PCBP protein family can function in PV RNA replication. In future studies, these evolutionarily related, but f unctionally distinct isoforms can be used to direct more detailed mutagenic st udies of the individual f unctional domains. This approach, in combination with the (MS2)2 protein-RNA tethering system, can then be used to more precisely define the protei n-protein interaction surfaces and binding partner critical to PCBPs ability to promote negative-strand synthesis.

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127 The (MS2)2 Protein-RNA Tethering System: Virus-Host Interaction A defining characteristic of a vi rus is its ability to commandeer its host cell and subvert the cellular machinery for its own replication. The (MS2)2 protein-RNA tethering system used in this study provides an ideal framework for additional studies on virus-host inte ractions critical to the understanding of virus replic ation and cellular protein func tions therein. The specific integration of key host proteins into defined steps in the viral life cycle relieves the need of the viral genome to encode such proteins, but also functions as a post-entry determinant in cell tropism. For viruses like poliovirus which inf ect multiple distinct cell types, these cellular protein determinants could function as replicative rheostats, allowing th e virus to tailor its replication to the cell type it ha s infected. These unique interac tions between vi ral and cellular proteins are also very attractiv e antiviral drug targets, particul arly given that cellular protein evolution is not subject to the same selective pressures as viral proteins. The (MS2)2 Protein-RNA Tethering Syste m: Host Protein Function An appealing extension to the (MS2)2 protein-RNA tethering system, in addition to the generalized study of host protein involvement in the replication of other RNA viruses, is the potential to better understand the no rmal cellular role of these key proteins. Viral systems serve as microcosms for complex host cell processes, and have provided the foundation for much of our current understanding of cellular biology. DNA replicat ion, mRNA splicing, innate immunity, endocytosis, oncogenesis, and apopt osis are among the many cellular processes initially characterized using viruses or viru s-based approaches. Likewise, by understanding precisely how key cellular protei ns are exploited during virus in fection, we can better understand their role in cellular processes and in the gl obal regulatory networks in which they often participate. This work would also extend to the ro le of these critical proteins in disease states, some of which may be directly related to virus inf ection. Dysregulation of PCBP regulated

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128 mRNAs has been linked to liver cirrhosis, cervical cancer, and cardiomyopathy (123, 169, 191, 198). Interestingly, each of these conditions can also result from in fection by a virus that utilizes PCBP during its replication: Hepatitis C Vi rus (HCV), Human Papillomavirus (HPV), and Coxsackievirus B (CVB), respectively. These all serve as examples of complex disease states where critical protein-protein and/ or protein-RNA interactions coul d be initially examined in a simplified context, using a virus or virus-ba sed system in combination with the (MS2)2 tethered function system. The Role of Viral Protein Precursors in the Initiation of P V Negative-strand Synthesis Modeling Formation of the PV RNA Replication Complex The second and third lines of investigation both deal with the cr itical role of distinct viral polyprotein precursors in the initi ation of poliovirus negative-stra nd RNA synthesis. Firstly, we exam ine the molecular basis for the re quirement of genomic translation in cis to promote efficient initiation of negativ e-strand synthesis. Using trans complementation assays, we showed that the activity of a pr otein precursor, rather than ph ysical ribosome transit, was responsible for the observed cis enhancement of negative-stra nd synthesis. Further, we identified the critical cis -acting precursor as 2BC-P3 and generated a model of replication complex formation which accounts for this requiremen t. This model is able to account for the previously observed coupling between genomic translation and RNA replication observed in infected/transfected cells, as well as other previously reported protein complementation studies from our lab. The last line of investigation also utilized trans complementation assays in combination with functionally defined mutants of the multifunctional viral precursor 3CDpro. Using this approach, we defined the functional polyprotein precursor of the active polymerase in the replication complex to be 3CDpro, however we showed that the pr eferred precursor utilized to

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129 form the essential 5CL RNP complex was P3. These experiments also validated the current model for initiation of negative-strand synthesi s, which depicts two functionally discrete 3CDpro polypeptides within the PV replic ation complex. This study allowe d us to enrich our model of PV replication complex formation to include greate r detail as to the source of protein precursors which form the critical 5CL RNP complex, pr ovide the VPg primer for RNA synthesis, and generate active 3Dpol. Close Coupling of the Viral Life -Cycle Ensures Viral F itness Both of these studies, in addition to defini ng critical components of the PV replication complex, also illustrate the tightly coupled natu re of viral replication. In most cases, complementation in trans of a viral protein supports significan tly lower levels of negative-strand RNA synthesis than would be observe d if that protein was provided in cis The evolutionary imperative to tightly couple the different stages of the viral life-cycle stems from the complexity inherent in coordinating a very intricate sequence of events in the context of the chaotic milieu of a host cell, in the face of extens ive innate anti-viral defenses. This task is only complicated further by the high mutation rates exhibited by R NA viruses and the need to counter-balance the increased speed of viral evolution with extensive genomic quality control. However, by doing this, a virus ensures replication of complete genomes encoding fully functional proteins to the exclusion of incomplete or def ective genomes, preventing the wast eful use of valuable cellular resources. Poliovirus, like other small RNA viruses, maximizes protein function using limited genomic sequence space by encoding a singl e large polyprotein and utilizing each unique precursor within the protein processing cascade. It now also appears th at polyprotein processing also contains within it the intrinsic ability to tightly couple cis translation of PV RNA and subsequent replication complex formation. This coupling functions as a critic al replication checkpoint, a penultimate guarantee that the PV template RNA about to be replicated encodes a

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130 functional set of essential re plication proteins, ensuring e fficient RNA replication and evolutionary maintenanc e of viral fitness.

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149 BIOGRAPHICAL SKETCH Allyn Russell Spear was born in Milwaukee, W isconsin in August of 1981 to Neal and Marlyn Spear. He grew up in Wauwatosa, Wisc onsin and graduated from Wauwatosa East High School in June 1999. Following this, Allyn attended the University of Wisconsin-La Crosse, and graduated in May 2003 with bachelors degrees in both microbiology and chemistry with an ACS certification. While at the University of Wisconsin-La Crosse Allyn had the opportunity to train under the direction of Dr. Michael A. Hoffman, performing research on the role of the matrix protein in the assembly and budding of human parainfluenzavirus type-3. In August 2003, Allyn began the Interd isciplinary Program in Biomedical Sciences at the University of Florida. In the Spring of 2004, he had the opportunity to begin doctoral research in the laboratory of Dr. James Bert Flanegan, studying the biochemistry and molecular biology of poliovirus replication. Under Dr. Flanegans di rection, Allyn completed all required coursework and dissertation research in the Summer of 2009.