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Regulation of the Herpes Simplex Virus Type-1 (HSV-1) Latency-Associated Transcript (LAT)

Permanent Link: http://ufdc.ufl.edu/UFE0019662/00001

Material Information

Title: Regulation of the Herpes Simplex Virus Type-1 (HSV-1) Latency-Associated Transcript (LAT)
Physical Description: 1 online resource (101 p.)
Language: english
Creator: Giordani, Nicole V
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: hsv1, latency, reactivation, regulation
Immunology and Microbiology (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: Herpes simplex virus type 1 (HSV-1) establishes latency in neurons until stimulated by stress to reactivate. The latency-associated transcript (LAT) region is the only portion of the latent genome that is actively transcribed during latency. While the LAT region is known to facilitate reactivation, the exact mechanism involved is not known. The overall focus of this dissertation was to investigate elements in the LAT promoter that respond to stress and alter transcription in this regulatory region. Here, the chromatin profile of a reactivation-negative HSV-1 LAT promoter mutant, 17deltaPst, was assessed prior to and following adrenergic induction of reactivation in the rabbit ocular model. In contrast to the latent chromatin profile of wild-type 17syn+, 17deltaPst showed increased enrichment of dimethyl H3 K4 just upstream of the deleted region, suggesting that deletion of the LAT promoter causes a loss of a regulatory element required for initiation of reactivation. Post-induction of reactivation, no apparent chromatin remodeling occurred for either virus during early times (1, 2, 4 hours). To investigate a possible regulator of the LAT promoter, a cAMP response element, located 83 nucleotides upstream of the LAT transcriptional start site (-83CRE) was examined. A recombinant virus with mutation of the -83CRE was created and analyzed for alterations to the acute, latent, and reactivation phases of the viral lifecycle. The -83CRE recombinant was found to have a second site mutation somewhere in the genome. During acute infection, the -83CRE recombinant displayed attenuated virulence in mice when infected via the footpad, and in cell culture the virus showed increased replication in epithelial cells and decreased replication in neuronal cells, suggesting that the mutations in the -83CRE recombinant play a role in viral replication. The -83CRE recombinant virus was also found to establish latency, express LAT, and reactivate from latently-infected rabbits. These results indicate that the LAT promoter contains elements that regulate transcription at the level of chromatin, which may play a key role in facilitating stress-induced reactivation. In addition, data from analyses of the -83CRE recombinant suggest that a compensatory mutation generated during viral construction plays a role in virulence and may interact with the -83CRE site to regulate lytic infection. Overall, this study invokes a model where the LAT promoter acts as complex regulatory switch that modulates gene expression in a tissue-specific manner both during the acute and latent periods of HSV-1 infection.
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 Nicole V Giordani.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Bloom, David C.
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 2007
System ID: UFE0019662:00001

Permanent Link: http://ufdc.ufl.edu/UFE0019662/00001

Material Information

Title: Regulation of the Herpes Simplex Virus Type-1 (HSV-1) Latency-Associated Transcript (LAT)
Physical Description: 1 online resource (101 p.)
Language: english
Creator: Giordani, Nicole V
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: hsv1, latency, reactivation, regulation
Immunology and Microbiology (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: Herpes simplex virus type 1 (HSV-1) establishes latency in neurons until stimulated by stress to reactivate. The latency-associated transcript (LAT) region is the only portion of the latent genome that is actively transcribed during latency. While the LAT region is known to facilitate reactivation, the exact mechanism involved is not known. The overall focus of this dissertation was to investigate elements in the LAT promoter that respond to stress and alter transcription in this regulatory region. Here, the chromatin profile of a reactivation-negative HSV-1 LAT promoter mutant, 17deltaPst, was assessed prior to and following adrenergic induction of reactivation in the rabbit ocular model. In contrast to the latent chromatin profile of wild-type 17syn+, 17deltaPst showed increased enrichment of dimethyl H3 K4 just upstream of the deleted region, suggesting that deletion of the LAT promoter causes a loss of a regulatory element required for initiation of reactivation. Post-induction of reactivation, no apparent chromatin remodeling occurred for either virus during early times (1, 2, 4 hours). To investigate a possible regulator of the LAT promoter, a cAMP response element, located 83 nucleotides upstream of the LAT transcriptional start site (-83CRE) was examined. A recombinant virus with mutation of the -83CRE was created and analyzed for alterations to the acute, latent, and reactivation phases of the viral lifecycle. The -83CRE recombinant was found to have a second site mutation somewhere in the genome. During acute infection, the -83CRE recombinant displayed attenuated virulence in mice when infected via the footpad, and in cell culture the virus showed increased replication in epithelial cells and decreased replication in neuronal cells, suggesting that the mutations in the -83CRE recombinant play a role in viral replication. The -83CRE recombinant virus was also found to establish latency, express LAT, and reactivate from latently-infected rabbits. These results indicate that the LAT promoter contains elements that regulate transcription at the level of chromatin, which may play a key role in facilitating stress-induced reactivation. In addition, data from analyses of the -83CRE recombinant suggest that a compensatory mutation generated during viral construction plays a role in virulence and may interact with the -83CRE site to regulate lytic infection. Overall, this study invokes a model where the LAT promoter acts as complex regulatory switch that modulates gene expression in a tissue-specific manner both during the acute and latent periods of HSV-1 infection.
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 Nicole V Giordani.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Bloom, David C.
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 2007
System ID: UFE0019662:00001


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b2cbeaa7bfdcf612ee45fb2dee4fa601
69cc10e44d78ee21713b840632aedafccf72cf8c







REGULATION OF THE HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) LATENCY-
ASSOCIATED TRANSCRIPT (LAT)



















By

NICOLE V. GIORDANI


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

2007





































O 2007 Nicole V. Giordani









ACKNOWLEDGMENTS

First and foremost, I thank my parents and family for encouraging me to follow whatever

career path I chose. I wouldn't be where I am today without their support and love, and I will

always be grateful.

I thank my undergraduate mentor, Richard Hallick, for giving me the chance to get my

start in a lab. In addition, I thank his post-doctoral associate, Elena Sheveleva, for her patience

and kindness during her days of working with me.

I especially thank Dave for his guidance over the past few years. He has taught me a lot

about many aspects of science and has always provided encouragement, even when I was not

sure that I could do it. I have always appreciated the enthusiasm with which he approaches

science, and I hope some of it has worn off on me.

I thank all of the graduate students (Niki, Jerry, Anne, Lee, Tony, Zane, and Dacia) that I

worked with over the years for each teaching me something and helping me whenever I needed

it. To list what I have learned from each of them would be impossible, since the knowledge they

have passed on to me has been insurmountable. I also thank Dacia for critical reading of this

dissertation and for helping me to deal with the stress. I thank Peterj on ("the best tech ever") for

all of his computer and cell culture assistance and for helping me with so many other things here

and there. I thank Joyce for her expertise with cloning and for always offering to help when she

sees the work piling up.

I thank my committee--Jorg Bungert, Rich Condit, Bert Flanegan, and Dick Moyer-for

their helpful suggestions and for keeping me on track.

Many other people have helped shape me as a scientist, and I thank all of them.











TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............3.....


LIST OF TABLES ................ ...............7............ ....

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


AB S TRAC T ........._. ............ ..............._ 10...

CHAPTER


1 INTRODUCTION ................. ...............12.......... ......

Basic Overview............... ...............12
Productive Infection............... ...............1
Latency and Reactivation .............. ...............14....
Anim al M odels ................. ............ ............ .............1
The Latency-Associated Transcript (LAT) .............. ...............16....
The LAT during lytic infection ................ ...............17........... ...
The LAT during latency and reactivation .............. ...............17....
LAT Promoter and LAT Transcription .............. ...............19....
Eukaryotic Epigenetic and Transcriptional Regulation ................. ................ ......... .23
Epigenetic Regulation ............... ... ... .................2
Promoter Elements and Transcriptional Regulation............... ...............2
Summary ................. ...............27.................

2 CHROMATIN CONFORMATION OF THE LATENT HSV-1 GENOME INT
RABBIT S .............. ...............3 1....


Obj ective ................ ........ .... ...............3.. 1....
Background and Previous Findings ................. ...............31................
Materials and Methods .............. ...............34....
Rabbit Infections .............. ...............34....
ChlP A ssay .................... ... ...... ............3
Taqman Real-Time PCR Analysis .............. ...............35....
R esults...... ... ........ ....... ..... ........ .............. .................... .............3
The Chromatin Profile of the Latent HSV Genome in Rabbits Latently-Infected
with Wild-Type HSV-1 Is Similar to that Observed for Latently-Infected Mice........36
Deleting the Core LAT Promoter Results in Increased H3 K4 Dimethylation of the
LAT Promoter Region in Latently-Infected Rabbits ................... ..... ............3
Neither 17syn+ nor 17APst Display Dynamic Changes in the LAT Region in
Response to Epinephrine Induction of Latently-Infected Rabbits ............... ..............3 7
Relative H3 K4 Dimethylation Levels of the LAT Promoter Region in 17A~st Are
Higher than Those of Wild-type for All Times Examined ............_.. ........._.....37











D discussion .........._.... .... .... ..... ...............37..
Kinetics of Chromatin Remodeling ........._...... ...............37..__._. .....
A Repressive Element in the LAT Promoter ...._._._.. ..... ..__... ......__.. .........3

3 INVESTIGATION OF THE ROLE OF A LAT PROMOTER cAMP RESPONSE
ELEMENT (CRE) INT REACTIVATION .............. ...............44....


Obj ective ................ ........ .... ...............44.......
Background and Previous Findings ................. ...............44................
M materials and M ethods .............. ..... .. .... ..... .......4
Plasmid Generation, Mutagenesis, and Purification............... ..............4
Cells and Viruses ............... ... ...............50................
DNA Isolation for Transfections ................. ....__ ....___ ............5
Virus Construction and Plaque Purification .....__ ................ ............... 51.....
PCR Analysis............... ...............54
Growth Curves............... ...............54.
Mouse Survival As say .........__ ...... ...___ ...........__ .........5
Intracranial Inoculation as an Assay for Neurovirulence ................ .......................55
DNA Extraction and Analysis of Course of Infection. .................. ...... .......... ..............55
RNA Isolation and Reverse Transcription for Acute RNA Levels in Cell Culture ........56
Taqman Real-time PCR Analysis................... .......................5
RNA Isolation and Reverse Transcription for Explant Studies............... .................5
ChlP Analysis............... ...............58
Rabbit Reactivation .............. ...............59....
R e sults.................. ... ....... .. ....... ... ... ...... .. ....... .. ........6
The -83 CRE Recombinant' s Replication Is Altered during the Lytic Phase of the
Infection .................. ......... .. .. ......... .... .. ................61
The -83CRE Recombinant Displays Impaired Replication and Spread in the
Nervous System of the Mouse ........._...... ........._... ...............62....
The -83CRE Virus Contains a Second Site Mutation............. ...... ....................... 6
Mutation of the -83 CRE Results in Wild-type HSV RNA Levels in RS Cells during
Acute Infection............... ........... .............6
The -83 CRE Virus Expresses LAT during Latency .............. ........ ..._ ................65
The -83CRE Virus LAT Promoter Region's H3 K9, Kl4 Acetylation Levels Are
Similar to Those of ICPO during Latency ........._.............. ..._. ... .. ....._._.. ..........6
The -83CRE Virus Reactivates from Latency in the Rabbit with Similar Efficiency
as W ild-type 17syn+ ............ ...............66.....
D discussion ........._..._........ ....... ... .. ..... .. _... .. .... ... .. .......6
Mutation of the -83 CRE Is Unfavorable for Recombination ................ ......... ........... ....66
The -83CRE Recombinant Contains a Second Site Mutation that Contributes to the
Avirulence Phenotype ................ ... ... ............ .... ...... ..... ... .... ........6
Cell-Specific Factors May Interact with the -83CRE Site to Convey Neuronal
Tropism to HSV-1 ............... .. ...... ...... .. ....... ... .. ... .. ........7
Mutation of the -83 CRE Site Does Not Affect Latency and Reactivation.....................71
The Wild-Type -83CRE May Control LAT during the Acute Infection.........................73












4 OVERALL DI SCU SSION ................. ...............8.. 4......... ...


The LAT and Chromatin ............... ... .. ...............84
LAT Regulation through Promoter Function .............. ...............86....
A Model for Reactivation ........._.___..... .__. ...............87...


APPENDIX REAL-TIME PCR PRIMER/PROBE SEQUENCES............_._._ ........_._. .....90


LIST OF REFERENCE S ........._.___..... .___ ...............91....


BIOGRAPHICAL SKETCH ........._.___..... .__. ...............101....










LIST OF TABLES


Table page

3-1 Survival of mice infected via the footpad with either 17syn+ or -83 CRE .......................74

3-2 Survival of mice inoculated intracranially with either 17syn+ or -83CRE. .....................74

3-3 Slit lamp examination (SLE) scores of rabbit corneas at 3, 5, or 7 days post-infection.. ..74

3-4 Reactivation of rabbits post-epinephrine induction ................ .............................74

A-1 Real-time PCR primer/probes used .............. ...............90....










LIST OF FIGURES


FiMr page

1-1 Diagram of the H SV-1 virion ..........._ ..... ..__ ...............29.

1-2 Reactivation critical region (rcr) .............. ...............29....

1-3 Mutants in the reactivation critical region .............. ...............30....

1-4 Elements of the core LAT promoter ........... .....__ .. ...............3

2-1 Diagram of the chromatin profile of the latent HSV-1 genome in the mouse..................40

2-2 LAT region histone H3 K4 dimethylation status of latently-infected rabbits for
17syn+ or 17A~st ................. ...............40.......... ....

2-3 Dimethyl H3 K4 status during epinephrine-induced reactivation of 17syn+ ................... .41

2-4 Dimethyl H3 K4 status during epinephrine-induced reactivation of 17APst ................... ..42

2-5 Ratios of average relative H3 K4 dimethylation of 17APst to those of 17syn+ for
epinephrine-induction in rabbits .............. ...............43....

3-1 Diagram of PCR primer locations used in verification of -83 CRE mutant virus.............75

3-2 Analysis of PCR products amplified from dot blot purification ...........__..................75

3-3 Multi-step viral growth curves (m.o.i. of 0.01) for 17syn+ or -83CRE on RS cells,
Neuro-2A cell s, and L7 cell s .............. ...............76....

3-4 Single-step viral growth curves (m.o.i. of 5) for 17syn+ or -83CRE on RS cells and
Neuro-2A cell s ................. ...............77.......... ......

3-5 Percent survival over the course of acute infection of mice infected with either
17syn+ or -83CRE at 500 pfu, 5,000 pfu, or 50,000 pfu ................... ............... 7

3-6 Relative viral genomes for 8 hours, 2 days, or 4 days p.i. of mice infected via the
footpad with either 17syn+ or -83 CRE ........... _.....__ ...............79

3-7 Percent survival of mice infected with 500 pfu, 5,000 pfu, or 50,000 pfu of 17syn+,
-83CRE, or F8-1 rescuant virus. ............. ...............80.....

3-8 Multi-step growth curve (m.o.i. of 0.001) on RS cells for 17syn+, -83CRE, and F8-1 ....81

3-9 Relative RNA transcript levels during acute infection of RS cells with 17syn+ or
-83CRE recombinant .............. ...............82....











3-10 LAT expression during latency in mouse DRG infected with the -83CRE mutant
virus............... ...............83.

3-11 The LAT promoter of the -83CRE mutant virus is decreased in histone H3 K9, Kl4
acetylation relative to wild-type............... ...............8









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

REGULATION OF THE HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) LATENCY-
ASSOCIATED TRANSCRIPT (LAT)

By

Nicole V. Giordani

August 2007

Chair: David C. Bloom
Major: Medical Sciences--Immunology and Microbiology

Herpes simplex virus type 1 (HSV-1) establishes latency in neurons until stimulated by

stress to reactivate. The latency-associated transcript (LAT) region is the only portion of the

latent genome that is actively transcribed during latency. While the LAT region is known to

facilitate reactivation, the exact mechanism involved is not known. The overall focus of this

dissertation was to investigate elements in the LAT promoter that respond to stress and alter

transcription in this regulatory region.

Here, the chromatin profile of a reactivation-negative HSV-1 LAT promoter mutant,

17APst, was assessed prior to and following adrenergic induction of reactivation in the rabbit

ocular model. In contrast to the latent chromatin profile ofwild-type 17syn+, 17APst showed

increased enrichment of dimethyl H3 K4 just upstream of the deleted region, suggesting that

deletion of the LAT promoter causes a loss of a regulatory element required for initiation of

reactivation. Post-induction of reactivation, no apparent chromatin remodeling occurred for

either virus during early times (1, 2, 4 hours).

To investigate a possible regulator of the LAT promoter, a cAMP response element,

located 83 nucleotides upstream of the LAT transcriptional start site (-83CRE) was examined. A

recombinant virus with mutation of the -83 CRE was created and analyzed for alterations to the









acute, latent, and reactivation phases of the viral lifecycle. The -83CRE recombinant was found

to have a second site mutation somewhere in the genome. During acute infection, the -83CRE

recombinant displayed attenuated virulence in mice when infected via the footpad, and in cell

culture the virus showed increased replication in epithelial cells and decreased replication in

neuronal cells, suggesting that the mutations in the -83CRE recombinant play a role in viral

replication. The -83CRE recombinant virus was also found to establish latency, express LAT,

and reactivate from latently-infected rabbits.

These results indicate that the LAT promoter contains elements that regulate transcription

at the level of chromatin, which may play a key role in facilitating stress-induced reactivation. In

addition, data from analyses of the -83CRE recombinant suggest that a compensatory mutation

generated during viral construction plays a role in virulence and may interact with the -83CRE

site to regulate lytic infection. Overall, this study invokes a model where the LAT promoter acts

as complex regulatory switch that modulates gene expression in a tissue-specific manner both

during the acute and latent periods of HSV-1 infection.









CHAPTER 1
INTTRODUCTION

Basic Overview

The virus family Herpesvirid aedddd~~~~~ddddd is composed of a number of double-stranded DNA

(dsDNA) viruses that share patterns of gene expression and the ability to undergo latency, a state

in which the viral genome is virtually shutdown until reactivated (reviewed in Wagner and

Bloom, 1997). There are herpesviruses that infect a wide range of hosts, from mollusks to

mammals. In humans there are eight known herpesviruses, with different primary sites of

infection and cell types in which they can become latent. Herpes Simplex Virus type-1 (HSV-1)

is a member of the ot-herpesvirus subfamily, a group whose members--HSV-1, HSV-2, and

Varicella-Zoster Virus--have a tropism for neuronal cells during latency.

HSV-1 is very common in the population, with approximately 60-90% seropositivity in the

adult U.S. population (Smith and Robinson, 2002). While the virus usually only causes painful,

inconvenient cold sores, it can also cause herpes keratitis, the leading cause of infectious

blindness in the U.S. (reviewed in Biswas and Rouse, 2005), and in rare instances, can cause

encephalitis and even death (reviewed in Higgins et al., 1993). There is no cure for HSV-1, and

while drugs affecting specific viral targets during the periods of active replication (i.e., aciclovir

interferes with HSV-1 DNA replication) can limit productive infection, they are not effective

against the latent virus. Thus, by studying latency and reactivation, we can better our

understanding of HSV-1, which can eventually lead to better treatment options and possibly

yield a way to prevent reactivation.

Productive Infection

An infectious herpes virion consists of a glycoprotein-spiked envelope surrounding the

amorphous tegument, an icosahedral capsid, and the genome-containing core (Figure 1-1). The









HSV-1 genome is large when compared to other viruses (approximately 152 kilobases (kb)) and

encodes approximately 80 gene products during productive infection of the mucosal epithelium,

the usual site of entry. The expression of genes is temporally ordered, with immediate-early

genes being made before early genes which are made before late genes. Virions can also enter

sensory neurons, where the virus can enter latency as a nucleosome-associated, nonreplicating

episome (Rock and Fraser, 1985; Deshmane and Fraser, 1989) until stressing stimuli induce the

viral genome to reactivate from its quiescent state. At this time the virus can be transported by

anterograde axonal transport to once again cause productive infection at the epithelial surface.

The HSV-1 virion first enters its host cell through glycoprotein-receptor fusion, leaving

some tegument proteins, including viral host shutoff (vhs, which degrades cellular and viral

RNA), in the cytoplasm, while the nucleocapsid travels to the nucleus. At the nuclear pore, the

viral DNA is released into the nucleus. Accompanying the viral genome into the nucleus is the

tegument protein, VPl16, which interacts with cellular factors to enhance transcription of the

virus's immediate early genes.

Once the viral genome is in the host cell nucleus, the lytic cascade of transcription can

commence. Each class of genes must be transcribed in order for transcription of the next class to

begin. The first group of genes made is the immediate-early (IE or alpha) genes. There are five

of these genes (ICP4, ICPO, ICP27, ICP22, and ICP47), and this class provides the gene products

necessary for the expression of the next gene class, the early (E or beta) genes. The early genes

are crucial for replication and include polymerase, proteins for DNA and ORI binding, and the

helicase/primase complex. Expression of this class is down-regulated after the start of

replication of the viral genome. The final group of genes, the late (L or gamma) genes, provides

the more than 30 gene products coding for structural components of the virion.










Once all classes of gene products have been made, the viral capsid assembles in the

nucleus and buds through the inner nuclear membrane, acquiring the tegument layer and an

envelope. The virus particle is de-enveloped at the outer nuclear membrane and reacquires an

envelope at the Golgi apparatus. Once the mature virion is made, virions are spread by cell-to-

cell fusion, or they are released from the cell by exocytosis to initiate a second round of

infection.

Latency and Reactivation

Animal Models

Studies performed in cell culture have provided much information about the biology of

HSV-1, particularly molecular details of gene regulation during the acute infection. However,

due to the virus's ability to undergo latency in neurons, studies in cell culture have been limited.

While several groups have managed to induce a shut-down state of the viral genome in cell

culture (O'Neill et al, 1972; Preston et al, 1991; Moriya et al, 1994), none have been shown to

sufficiently mimic in vivo latency, so the most relevant means to study HSV-1 latency and

reactivation is through the use of animal models. Both the mouse and the rabbit have been

invaluable to the advancement of the understanding the latent phase of HSV-1 infection.

Mouse footpad model. There are several models of latency and reactivation that employ

the mouse. Once the mouse is infected through the eye, footpad or other route of epithelial

inoculation, the virus will establish a latent infection within the sensory ganglia that enervate the

region of primary infection. Reactivation is most often induced by hyperthermia or explant of

ganglia. Clinical reactivation or virus shedding at the site of initial inoculation does not

reproducibly occur, thus placing a limitation on the relevance of the mouse models to human

infection. However, mouse systems are more relevant than cell culture, are cost-efficient, and










while reactivation occurs in an in vitro manner, the mouse provides useful insight into the

molecular events related to latency and reactivation.

For the purposes of this dissertation, of the existing mouse models, only the mouse footpad

model has been used for the experiments herein, and therefore, this will be the only mouse model

for latency and reactivation that is discussed; this is not intended to trivialize the other murine

systems but only to provide the background for later discussion.

In the mouse footpad model, the keratinized epithelia of the rear footpads are first softened

by inj section of saline. The footpad is then abraded with an emery board, allowing a direct route

for viral entry into the anesthetized animal. The virus replicates in the footpad and then enters

the peripheral nervous system, traveling to the dorsal root ganglia (DRG), the spinal cord and

even the brain. If inoculated with a virulent strain of HSV-1, mice succumb to viral encephalitis

at approximately day 7 to 10 post-infection. In surviving mice the virus becomes latent in

neurons of the DRG, and after 21 to 28 days, there is no longer evidence of acute infection (lytic

viral transcription has ceased, the LAT is expressed, and infectious virus is no longer present).

At this time the DRG can be dissected from the mouse, and the latent state can be assessed or the

DRG can be explanted to supplemented media for a given length of time, in order to examine

molecular events that occur in response to stress. Additionally, the latently-infected DRG can be

co-cultivated on a cell monolayer to analyze the kinetics or efficiency of explant-induced

reactivation.

While these methods are not considered to necessarily mirror in vivo or clinical

reactivation, DRG explant still provides useful information and can serve as a starting point for

investigation of this process.









Rabbit eye model. In human HSV-1 infection, latency occurs in the trigeminal ganglia

(TG), and reactivation occurs at the site of primary infection, commonly the mucosal epithelia.

This is similar to what is observed in the rabbit eye model--rabbits are infected through the

corneas, latency occurs in the TG, and upon reactivation infectious virus can be recovered at the

site of initial infection. In addition to spontaneous reactivation, it is possible to induce

reactivation in latently-infected rabbits. Iontophoresis of epinephrine via a direct current to the

eye has been shown to reliably induce viral shedding from latently-infected rabbits at high

frequencies (Kwon et al., 1981). Because epinephrine is a hormone released in response to

stress, the use of it seems quite relevant. In fact, when rabbits are administered propanolol, a

blocker of the P-adrenergic receptors that bind epinephrine, there is a significant decrease in the

levels of spontaneous reactivation (Kaufman et al., 1996). At the present time, the rabbit eye

model is arguably the most relevant model for reactivation studies.

The Latency-Associated Transcript (LAT)

When HSV-1 becomes latent in a neuron, there is an overall shutdown of the genome, with

the exception of the latency-associated transcript (LAT) region (Stevens et al., 1987). The LAT

is approximately 8.3-8.5-kb in length (Dobson et al., 1989; Mitchell et al., 1990), and the

primary transcript can be spliced to yield 2.0- and 1.5-kb species (Wagner et al., 1988; Wechsler

et al., 1988). The 2.0-kb LAT has been demonstrated to be a stable intron (Farrell et al., 1991),

with a half-life of almost 24 hours in cell culture (Thomas et al., 2002), while the smaller 1.5-kb

appears to be a smaller splice product of the 2.0-kb intron produced only in a subpopulation of

neurons that express the LAT. Furthermore, studies performed with a transgenic mouse

containing the LAT promoter through the 2.0-kb intron demonstrated that while the transgene

was expressed in various tissue types, high levels of 2.0-kb intron accumulation occurred only in









sensory ganglia and appeared to be differentially spliced in those tissues (Gussow et al., 2006).

While it seems likely that the 2.0-kb LAT intron is important to the virus when in sensory

ganglia, no precise function has been ascribed to the LAT splice products, and the 2.0-kb intron

is dispensable for normal reactivation (Jarman et al., 2002).

The LAT during lytic infection

While the LAT is the prominent transcriptional unit during latency, LAT is also detectable

in murine TG during lytic infection (Spivack and Fraser, 1988) and is expressed as a late gene in

cell culture (reviewed in Wagner et al., 1995). In addition, the LAT promoter is active in both

neuronal and non-neuronal mouse tissue during the lytic phase of infection (Jarman et al., 1999),

suggesting that simply expressing LAT is not enough to cause latency. Finally, one study

demonstrated that in acutely-infected murine ganglia, the virus may simultaneously follow

productive and latent pathways, the former showing expression of lytic genes and decreased

LAT levels and the latter displaying almost no viral transcription except for the LAT (Margolis

et al., 1992). The study also demonstrated that the two pathways occur in two different subsets

of neurons, which likely drives the outcome of the infection into being productive or latent

(Margolis et al., 1992).

The LAT during latency and reactivation

The exact role of the primary LAT in latency and reactivation is still unknown. Promoter

deletion mutants that do not express the LAT retain wild-type levels of establishment and

maintenance of latency (Ho and Mocarski, 1989; Javier et al., 1988; Sedarati et al., 1989; Steiner

et al., 1989), arguing against a critical role for the LAT in those functions. In contrast, however,

establishment of latency by a 1.9-kb deletion mutant of the LAT promoter through the 5' exon

was reduced by approximately 75% relative to the parental and rescued virus; a significantly

higher number of neurons in the TG were destroyed by the mutant than by the wild-type virus,










suggesting a role for the LAT in neuronal survival (Thompson and Sawtell, 2001). Neuronal

survival and anti-apoptotic activities have been attributed to the LAT in several studies (Perng et

al., 2000; Inman et al., 2001; Ahmed et al., 2002; Jin et al., 2003), and furthermore, a link

between spontaneous reactivation and anti-apoptotic activity has been suggested (Jin et al.,

2003). It has also been recently suggested that the LAT 5' exon region encodes a microRNA

that regulates apoptosis, in order to permit reactivation of the virus, although the functionality of

this microRNA has yet to be demonstrated in the context of an HSV-1 infection (Gupta et al.,

2006).

A clear role for the LAT appears to be in reactivation. Promoter deletion mutants that do

not express LAT display inefficient in vivo reactivation when assessed in the rabbit (Hill et al.,

1990; Bloom et al., 1994; Perng et al., 1994). Additionally, through analysis of deletion mutants,

it was demonstrated initially that the region necessary for reactivation (reactivation critical

region, rcr) lies in the first 1.5-kb of the LAT (Bloom et al., 1996; Perng et al., 1996). This

region was further mapped to the first 699 bp of the LAT, after mutants containing 2.0-kb intron

deletions displayed wild-type levels of induced reactivation (Jarman et al., 2002). Thus, the

region of the LAT that is imperative for normal reactivation extends from the core LAT

promoter through the 5' exon (Figure 1-2). Additionally, this region has also been demonstrated

to possess both enhancer and long-term expression functions that allow the LAT promoter to

remain highly active throughout latency (Lokensgard et al., 1997; Berthomme et al., 2001).

Several studies have been performed in order to further map the rcr. These have involved

the analysis of mutants with subdeletions in the 5' exon region, and several of interest will be

discussed here. Mutant 17ASty contains a 370-bp deletion of most of the 5' exon (Figure 1-3).

This mutant generates the 2.0-kb intron, has normal replication kinetics, and displays a normal









level of recovery when latently-infected mice TG are co-cultivated (Maggioncalda et al., 1994).

In contrast, 17ASty shows an approximately 40% reduction in epinephrine-induced reactivation

of the rabbit (Hill et al., 1996). This demonstrates the importance in validating work performed

in the in vitro mouse model in the more in vivo rabbit eye model. Strangely, in contrast to the

reduction in induced reactivation of rabbits by the deletion mutant, spontaneous reactivation does

not appear to be altered from wild-type levels when that region is deleted (Perng et al., 1996b).

This suggests that there may be more than one pathway for reactivation--one that is stress-

responsive and dependent on some element within the 5' exon and one that occurs spontaneously

and does not require the region.

To further address the role of the 5' exon region in reactivation, mutant 17A348 was

studied. This mutant contains a 348-bp deletion that is located 217-bp downstream of the LAT

transcriptional start site (Figure 1-3). 17A348 expresses LAT and establishes a latent infection in

rabbits at a level similar to that of wild-type, but like 17ASty, this mutant displays a significantly

decreased level of epinephrine-induced reactivation (Bloom et al., 1996). Surprisingly, when

mutants containing overlapping subdeletions of the 348-bp region (Figure 1-3) are tested in the

rabbit, levels of induced reactivation are near that of wild-type (Bloom et al., 1996;

Bhattacharj ee et al., 2003). This suggests that there are multiple cis elements in the region that

play a role in reactivation and several must be deleted in order to detectably alter reactivation.

The region is clearly quite complex, and there may be different means for the virus to reactivate,

possibly requiring interplay between various components.

LAT Promoter and LAT Transcription

Wild-type expression of the LAT-and therefore, wild-type reactivation in the rabbit--is

dependent upon the presence of a 202-bp core promoter (Dobson et al., 1989). In fact, when the

core LAT promoter is deleted from wild-type 17syn+ to generate 17APst, epinephrine-induced









rabbit reactivation (recovery of infectious virus) is significantly decreased, from approximately

79% of 17syn+-infected rabbits positive for infectious virus to about 13.5% of 17APst-infected

rabbits positive (Bloom et al., 1994). The difference is not as dramatic when the DRG or TG are

explanted from the mouse to cell culture. Approximately 50% of 17APst-infected ganglia

reactivate (cause visible cytopathic effect when explanted to a cell monolayer) while nearly

100% of 17syn+-infected ganglia reactivate (Devi-Rao et al., 1994). This suggests that the

presence of the core LAT promoter may have a slight effect on explant-reactivation but appears

to be more important for epinephrine-induced reactivation in the rabbit. While this is likely due

to a function of the LAT, it may also be due to a cis-acting element contained within the region.

Within the 202-bp of the core LAT promoter are several transcription factor binding sites

that appear to be important for the full function of the core LAT promoter. Among the identified

sites are a TATA box (Dobson et al., 1989), two cAMP response elements (CREs) (Leib et al.,

1991; Kenny et al., 1994), and binding sites for the upstream stimulatory factor (USF)

(Zwaagstra et al., 1991; Kenny et al., 1997) and ICP4 (Batchelor et al., 1994) (Figure 1-4).

The TATA box is necessary for normal function of the LAT promoter. When

chloramphenicol acetyltransferase (CAT) assays were performed using a plasmid in which the

TATA box was mutated or deleted, a reduction in promoter activity was observed relative to

wild-type (Rader et al., 1993; Ackland-Berglund et al., 1995). In vitro transcription assays also

demonstrated that the TATA box is required for full LAT expression (Soares et al., 1996). In

addition, it was demonstrated that co-cultivation of cells with murine TG latently-infected with a

TATA box mutant virus yielded normal levels of reactivation via this method, even though the

mutant displayed significantly decreased levels of LAT expression, as assayed by in situ









hybridization (Rader et al., 1993). In other words, the ability of HSV-1 to express LAT does not

necessarily correlate with the ability to reactivate from explanted murine ganglia.

The CRE identified in the LAT promoter located approximately 43 base pairs upstream of

the LAT transcriptional start site (referred to here as -43CRE) (Leib et al., 1991) was

demonstrated to bind the CRE-binding type 1 (CREB-1) protein (Millhouse et al., 1998), a

known stress-responsive transcriptional activator. When the -43CRE was deleted and its

promoter activity was assayed by CAT assay, a decrease of three- to four-fold relative to wild-

type was observed (Ackland-Berglund et al., 1995), even though LAT expression was not

affected when assayed by ribonuclease protection assay or by in situ hybridization (Rader et al.,

1993; Ackland-Berglund et al., 1995). It was therefore suggested that the -43CRE has an

inducible rather than basal activity in the context of the LAT promoter (Ackland-Berglund et al.,

1995). Interestingly, the spacing of the -43CRE site relative to the TATA box appears to play a

role in activity. Insertion of 10 nucleotides between the CRE and the TATA box of the LAT

promoter resulted in 2-3 fold more CAT activity compared to that of the wild-type promoter,

while removal of 5 nucleotides decreased activity by 6-8 fold relative to wild-type; since the 10

bp spacing reflects a turn of the DNA helix, this suggests a requirement for interaction between

factors binding to the two elements (Ackland-Berglund et al., 1995).

Because the cAMP response pathway is activated in response to binding of epinephrine to

cell receptors, it seems likely that the CREs in HSV-1 play some role in reactivation. However,

when the -43CRE site deletion mutant was tested in the rabbit, epinephrine-induced reactivation

was intermediate between wild-type strain 17syn+ and promoter-deletion mutant 17A~st (Bloom

et al., 1997), suggesting that the -43CRE was not the only factor affecting reactivation.

Interestingly, LAT expression during latency was similar between the -43CRE mutant virus and









wild-type (Bloom et al., 1997). This may indicate that the ability to express the LAT does not

necessarily correlate with reactivation, but instead, elements within the LAT promoter may

interact with cellular and viral factors to yield wild-type levels of reactivation.

A second CRE was identified between nucleotides -75 to -83 relative to the LAT

transcriptional start site (-83CRE) (Kenny et al., 1994). This -83CRE was demonstrated to bind

a repressive form of CRE binding (CREB) protein, CREB-2 (Millhouse et al., 1998), suggesting

that this site may play a role in transcriptional repression of LAT. Other findings and

speculations on the role of the -83CRE will be discussed further in Chapter 3.

In addition to the CREs, a site capable of binding USF is present in the LAT promoter

(Zwaagstra et al., 1991; Kenny et al., 1997). USF binds to the E-box of a promoter and interacts

with transcriptional machinery as well as with chromatin remodeling proteins (reviewed in Corre

and Galibert, 2005). In bovine leukemia virus (BLV), gene expression of the 5' long terminal

repeat appears to be regulated by the exclusion of CREB from a CRE that overlaps an E-box;

mutation of the E-boxes appears to increase binding of CREB complexes to the CRE and also

increases gene expression (Calomme et al., 2004).

Through electrophoretic mobility shift (EMS) assay, it was demonstrated that the HSV-1

LAT promoter E-box can bind either of the two forms, USF-1 and USF-2 (Kenny et al., 1997).

When the -43CRE and E-box of the HSV-1 LAT promoter were mutated simultaneously and

examined in an in vitro transcription assay, transcription levels appeared to decrease more than

when either element was mutated alone, suggesting an interplay between the two (Soares et al.,

1996). Neither the potential interactions between the E-box and the -83CRE, nor the effects of

these elements on reactivation or LAT transcription in vivo have been investigated.










The LAT promoter, in addition to various transcription factor binding sites, also has a site

to which ICP4 can bind (Batchelor et al., 1994). When this ICP4-binding site is mutated to

abrogate binding, in vitro expression of the promoter occurs at aberrant times, displaying early

gene expression, as opposed to the usual late gene expression kinetics (Rivera-Gonzalez et al.,

1994). In other words, it appears that the ICP4 site may act to control LAT expression at

inappropriate times, such as during productive infection and reactivation.

In summary, the core LAT promoter possesses several binding sites that appear to play

some role in regulation of the LAT. It is possible that some of these function in certain cell types

and at certain times during the infection to control the region. Additionally, various studies

suggest interplay between different elements, indicating that this region is both complex and

important.

Eukaryotic Epigenetic and Transcriptional Regulation

HSV-1 gene regulation shares similarities with that of eukaryotes, including an association

with nucleosomes during latency (Deshmane and Fraser, 1989) and various eukaryotic

transcription factor binding sites throughout the genome. While there are other similarities, for

the sake of brevity, only the relevant aspects of epigenetics to HSV-1 transcriptional regulation

will be discussed here.

Epigenetic Regulation

Epigenetics can be defined as a modification in gene expression or cellular phenotype that

does not change the actual DNA (reviewed in Goldberg et al., 2007). More specifically, protein

interactions with DNA are capable of causing changes in gene expression. In order for DNA to

readily fit into cells, the genome is compacted into chromatin fibers, which is generally grouped

into two different classes, euchromatin and heterochromatin. Euchromatin is a more relaxed

structure, which allows access to the DNA by a range of transcription factors and other various










proteins, while heterochromatin is condensed chromatin with DNA that is inaccessible to

transcription factors and is therefore transcriptionally inactive. The basic unit of chromatin, both

euchromatin and heterochromatin, is the nucleosome, which consists of 147 bp of DNA wrapped

around an octamer of four core histones (H2A, H2B, H3, and H4) (reviewed in Kouzarides,

2007). Interactions of nucleosomes and DNA can be altered by three mechanisms: post-

translational modifications (PTMs), replacement with histone protein variants, and ATP-

dependent chromatin remodeling (reviewed in Bernstein and Hake, 2006). However, since

PTMs are more relevant than the others to the work in this dissertation, they will be discussed

here, and while a variety of PTMs have been characterized, only those that are most pertinent to

the work in this dissertation will be reviewed.

The addition of PTMs to the N- or C-terminal tails of histones can alter the transcriptional

permissivity of the nucleosomes. Histone H3 acetylation of lysine residues 9 and 14 (K9, Kl4)

and dimethylation of lysine residue 4 (K4) are both traditionally associated with regions of active

transcription (reviewed in Li et al., 2007). A yeast microarray study found that while histone H3

K4 dimethylation was not globally correlated with promoter regions of transcriptionally active

genes, there was a statistically significant association between the modification and the coding

regions of active genes (Bernstein et al., 2002). In contrast, the study also demonstrated that H3

K9, Kl4 acetylation was associated with the promoters of active genes, as well as within coding

regions, although to a slightly lesser extent. A separate study performed using genome scanning

of two human cell lines revealed that of 57 active genes analyzed, 58% displayed enrichment in

histone H3 K4 dimethylation and H3 K9, Kl4 acetylation within 500 bp of transcriptional start

sites, while 28% were farther down in the coding region (Liang et al., 2004). Thus, while there

may be subtle distinctions between where the PTMs are associated, it is commonly accepted that









histone H3 K9, Kl4 acetylation and histone H3 K4 dimethylation are markers of transcriptional

activity .

Chromatin studies performed on HSV-1 have focused mainly on active marks of

chromatin. During lytic infection of cell culture, it was shown that while the DNA is in a

partially nucleosomal state, association of the active histone mark, acetylated H3 K9, with viral

DNA occurs by one hour post-infection (p.i.) for ICPO, thymidine kinase, and VPl6 (Kent et al.,

2004). However, contrary to that study, Herrera and Triezenberg (2004) demonstrated that very

little histone H3 (nonacetylated or acetylated) is present at the IE gene promoters examined

(ICPO, ICP4, ICP27) during early lytic infection (2 h.p.i.), while the thymidine kinase, VPl6 and

glycoprotein C promoters are associated with acetyl-H3 K9, Kl4. One interesting difference in

these lytic infection experiments is that the former, in which acetylated H3 K9 was associated

with viral genes of all classes by 1 h.p.i., was performed in the neuronal SY5Y cell line, while

the latter was performed in the more epithelial-like HeLa cells. Differential chromatin patterns

may indicate that the chromatin conformation, including the association with histone H3, of the

HSV-1 genome differs between cell types, possibly impacting establishment of latency.

The ability of HSV-1 to exist as a repressed episome during latency suggests that viral

repression may occur at the level of chromatin. In fact, one study suggested that expression of

the LAT may cause increased levels of H3 K9 dimethylation and decreased levels of H3 K4

dimethylation of lytic gene promoters during murine infection (Wang et al., 2005). When the

LAT region is examined during latency, increased levels of H3 K9, Kl4 acetylation are observed

for the LAT promoter and 5'exon/enhancer regions relative to lytic genes (Kubat et al., 2004a;

Kubat et al., 2004b). Further, explant reactivation of murine DRG appears to induce dramatic

changes at early times in both the LAT region's and ICPO's transcriptional permissiveness as










assayed by acetyl-H3 K9, Kl4, whereby the LAT region seems to decrease in acetylation before

ICPO can begin to increase (Amelio et al., 2006). Thus, the dynamic regulation of chromatin

modifications appears to also impact the HSV-1 genome' s transcriptional permissiveness at

various stages during infection.

Promoter Elements and Transcriptional Regulation

In most eukaryotic cells, an active gene promoter contains a nucleosome-free region (NFR)

approximately 150 bp in size surrounding the core promoter (reviewed in Heintzman and Ren,

2006). After the region is hyperacetylated, chromatin remodeling occurs and histone-DNA

contacts are lost or nucleosome-unfolding takes place (Boeger et al., 2003; Reinke and Hoirz,

2003). This allows for binding and stabilization of the transcriptional machinery to the promoter

(reviewed in Heintzman and Ren, 2006).

While there are three types of RNA polymerases--I, II, and III--RNA polymerase II (Pol

II) is responsible for transcription of mRNA and other regulatory RNAs and will therefore be

discussed here. Transcription can initiate once Pol II is recruited to a gene's core promoter,

which surrounds the transcriptional start site and encompasses 70-80 surrounding base pairs that

are recognized by the transcriptional machinery, but the surrounding sequences may be part of

the proximal promoter, conveying tissue-specifieity or acting as a transcriptional enhancer

(reviewed in Heintzman and Ren, 2006). There is much variability in the factors that are bound

to a specific promoter, but promoters generally function in a similar manner. First, chromatin

remodeling allows Pol II and other transcription factors to gain access to the promoter; this

preinitiation complex (PIC), once properly positioned, melts the 1 1-15 bp of DNA around the

transcriptional start site for correct interaction of the Pol II with the DNA and then begins

transcription (reviewed in Heintzman and Ren, 2006).









Binding factor recruitment is essential for transcriptional activation, but proteins that bind

to a promoter may instead function to repress the gene. While active transcriptional repressors

target a gene at the level of chromatin, passive repressors can compete with transcriptional

activators for binding, bind to activators as inactive heterodimers to inhibit transcription, or bind

to coactivators to prevent activation of transcription factors (reviewed in Thiel et al., 2004). One

example of a transcriptional repressor is the inducible cAMP early repressor (ICER), which has

been suggested to play a role in HSV-1 reactivation through repression of LAT (Colgin et al.,

2001). ICER is expressed from the same locus as CREB, a transcriptional activator, but is

transcribed from an intronic promoter and does not contain an activation domain (reviewed in

Mayr and Montminy, 2001). ICER levels peak at 2-6 hours after cAMP stimulation, and ICER

prevents CREB from binding to CRE-containing promoters, including the promoter which drives

ICER' s own transcription (reviewed in Mioduszewska et al., 2003). Numerous other examples

of transcriptional repressors exist in eukaryotes and in conjunction with transcriptional

activators, can allow promoters to function as switches for transcription.

Summary

Many questions still remain regarding the HSV-1 LAT' s role in regulation of the viral

lifecycle. Numerous experiments have demonstrated that one maj or function appears to be in

reactivation, since the LAT promoter and the 5' exon are critical to wild-type reactivation. Other

activities have also been ascribed to the region, and a picture is emerging in which LAT may

play a larger regulatory role, with some of the various phenotypes attributed to LAT mutants

being secondary effects of this regulatory function. Clearly, understanding how LAT is

regulated at the level of transcription and in different cell types may provide insight into its

larger role on HSV biology. Experiments detailed in this dissertation were aimed to address

some of the questions regarding LAT regulation-specifically, (1) what is the importance of









histone tail modifications in the ability to reactivate from latency, and (2) does a stress-

responsive CRE in the LAT promoter play a regulatory role in reactivation? The answers to

these questions will hopefully provide some indication of the complex regulation of the HSV-1

LAT region, as well as open up avenues for future work.










SEnvelope

Tegument

Capsid

Core


Figure 1-1. Diagram of the HSV-1 virion. The envelope, tegument, capsid, and core are
indicated.


Reactivation critical region



5' EXON 2.-b NRO EXON



Figure 1-2. Reactivation critical region (rcr). The minimal region, mapped by various
subdeletions, that is necessary for wild-type in vivo reactivation in response to stress
is designated by the blue bar. The core LAT promoter (LAPl), shown in yellow, is
defined here as the promoter region encompassed by the two PstI restriction enzyme
sites. The LAT enhancer, extending through the 5' exon, is shown in red. Note that
this is not to scale.












R, R, U Rs


Bz
c-a


-Normal reactivation
SDecreased epinephrine-induced
reactivation


I


RL


(_(~(t_^~_(_~_~^~_~~


8
J

x

$


8-
-O
8 M,


17A1Pst
17 C y
17A21"~
17I201
17A.207
17Al10-
17191-
178116-


Figure 1-3. Mutants in the reactivation critical region. Normal reactivation, shown in black,
refers to the ability to reactivate at wild-type levels in the in vivo rabbit reactivation
model. Red bars indicate decreased reactivation.


IR I


TR ,


Binding m





Figure 1-4. Elements of the core LAT promoter (defined as the region encompassed by the two
PstI restriction enzyme sites).









CHAPTER 2
CHROMATIN CONFORMATION OF THE LATENT HSV-1 GENOME IN RABBITS

Objective

Preliminary work in the mouse model demonstrated that the HSV-1 genome is associated

with specific histone modifications during latency and that there are changes in chromatin

permissiveness that occur during explant induced reactivation. The goals of the studies

performed here were to investigate patterns of histone modifications in the rabbit, a more

relevant model for HSV reactivation and to (1) determine if the histone modifications observed

during latency in mice are conserved in the rabbit eye model, (2) determine if epinephrine-

induced reactivation results in the same pattern of chromatin changes as observed in the mouse

during explant, and (3) determine if the chromatin profile of a reactivation-negative mutant

differs from wild-type during latency and/or reactivation (this could suggest that the defect in

reactivation is related to inappropriate chromatin configuration affecting promoter accessibility

and/or transcriptional permissiveness).

Background and Previous Findings

HSV-1 is maintained as a nucleosome-associated episome during latency (Deshmane et al.,

1989). This observation suggested that insight into the transcriptional status and regulatory

framework could be determined by analyzing the specific histone modifications that are

associated with the different regions of the HSV genome. In order to analyze histone

modifications of the latent and reactivating HSV-1 genome, the chromatin immunoprecipitation

(ChIP) assay has been used. In the ChlP assay (reviewed in Kuo and Allis, 1999), histones are

crosslinked to DNA using formaldehyde, and these complexes are sonicated into fragments of

approximately 500-1000 bp. Histones are immunoprecipitated with the antibody of choice and

then de-crosslinked from the DNA. The freed DNA is then purified and analyzed by PCR.









As discussed in Chapter 1, there are two primary animal models used for the study of

HSV-1 latency and reactivation, the mouse and the rabbit. The mouse footpad model has

enabled much of the work related to epigenetic studies of the HSV-1 genome during latency and

has also provided some insight into chromatin remodeling that occurs during early DRG explant-

induced reactivation.

Using the mouse footpad model, it was determined that DNA methylation does not play a

role in the repression of the latent HSV-1 genome (Kubat et al., 2004). Instead, since the latent

genome is associated with nucleosomes (Deshmane et al., 1989), histone tail modifications

appear to provide some indication as to transcriptional permissiveness (Kubat et al., 2004).

Specifically, during latency the LAT promoter region is 2-3.5 fold more enriched in acetylated

histone H13 K9, Kl4, a marker of transcriptional permissiveness, than the lytic genes, ICP27 and

ICPO (Kubat et al., 2004). Upon further assessment of the LAT region--specifieally the

enhancer located in the 5' exon--it was observed that the enhancer region is more acetylated

(approximately 3.5 fold) than the LAT promoter, while lytic genes exist in a hypoacetylated, or

less transcriptionally permissive, state (Kubat et al., 2004). The same effect was seen for a LAT

promoter deletion virus, which makes no detectable LAT. With this mutant the LAT enhancer

was still hyperacetylated relative to the LAT promoter, which was still more acetylated than the

nearby lytic genes (Kubat et al., 2004). In summary, this previous work indicates that the LAT

region is maintained in a transcriptionally permissive state independent of LAT transcription,

while the lytic gene regions of the HSV-1 genome exist in a less transcriptionally permissive

state during latency (Figure 2-1).

The Eindings for the latent genome's chromatin configuration were extended to the mouse

explant model, which provides information about the molecular events in reactivation. The










question addressed was whether specific regions of the HSV genome undergo chromatin

remodeling in response to explant-induced stress. If so, this could indicate what key changes

occur early in reactivation. Latently-infected DRG were removed to media for times ranging

from 0 hours post-explant (h.p.e) to 4 h.p.e. and then processed for ChlP. The LAT 5'

exon/enhancer displayed at least a five-fold decrease in H13 K9, Kl4 acetylation occurring within

the first hour of explant, while the ICPO promoter exhibited an increase in acetylation between 2

and 3 h.p.e. (Amelio et al., 2006). This study additionally found that there was a dramatic

decrease in LAT RNA abundance between 2 and 3 h.p.e. (Amelio et al., 2006). Overall, these

findings suggest that there is a remodeling of the LAT region during early explant, whereby both

deacetylation of the LAT enhancer and a decrease in LAT levels occur before the ICPO promoter

can become more acetylated.

The mouse explant model is limited by allowing only in vitro reactivation studies to be

performed. The type of reactivation obtained through DRG explant is relatively LAT-

independent, so molecular reactivation occurs regardless of whether a mutant virus does not

transcribe LAT. A more relevant model is the rabbit eye model. Epinephrine-induced

reactivation of the rabbit occurs in vivo, can produce clinical lesions and shed virus at the eye,

and reactivation is more LAT-dependent, in that LAT promoter deletion mutant are severely

reduced in reactivation relative to wild-type (Hill et al., 1990; Perng et al., 1994). It is for this

reason that many of the studies initially performed using the mouse footpad model are validated

in the rabbit. The experiments described here utilized the rabbit eye model to determine if the

remodeling events observed in the mouse also occur following adrenergic induction of

reactivation in the rabbit.












Rabbit Infections

One to 2 kg New Zealand White rabbits were infected and housed at the Louisiana State

University Health Science Center's Animal Facility. Each rabbit eye received topical

proparacaine-HCI anesthesthetic prior to corneal scarification. Rabbits were infected with either

17syn+ or 17A~st virus inoculum at 50,000 pfu/eye. At days 3, 5, and 7 post-infection (p.i.), the

infection of the rabbit eyes was monitored by slit lamp examination for the presence of dendrites

on the cornea.

After 28 days p.i., rabbits were sacrificed (latent time point) or epinephrine-induced prior

to sacrifice (reactivation time points). If epinephrine iontophoresis was performed, rabbits were

anesthetized with isoflurane, and a solution of 0.015% epinephrine was administered to the

rabbit eye for 8 minutes at 0.8 mAmps. At 0, 1, 2, or 4 hours post-induction, rabbits were

anesthetized with ketamine/xylazine and euthanized with a lethal dose of sodium pentobarbital.

After decapitation, rabbit trigeminal ganglia (TG) were removed and processed.

ChlP Assay

ChlP assays were performed at the University of Florida. Rabbit TG were homogenized in

0.5 ml phosphate-buffered saline (PBS) in the presence of protease inhibitors (1 Cpg/ml aprotinin,

1 Cpg/ul leupeptin, and 1 mM PMSF). DNA-histone complexes were crosslinked by the addition

of 37% formaldehyde to a final concentration of 1%. After the addition of 0. 128 M glycine, the

sample was pelleted and washed three times with PBS containing protease inhibitors as

described above. After the final wash, pellets were resuspended in SDS-lysis buffer (1% SDS,

10 mM EDTA, 50 mM Tris-HC1), and sonicated (Fisher Sonic Dismembrator 100) to yield

fragments of 500-1000 bp (Setting 4, 6 V/2 bursts of 40 sec. each). Sonicated samples were pre-

cleared with Salmon Sperm DNA/Protein A Agarose beads (Upstate), and histone-DNA


Materials and Methods









complexes were immunoprecipitated overnight with 3.5 Cpg/ml of anti-acetyl-Histone H3 K9/Kl4

(Upstate) or 1 Cpg/ml of anti-dimethyl-H3 K4 (Upstate). Prior to the wash steps, 25% of the

sample was removed and retained as the "unbound" fraction. Complexes were washed with Low

Salt (0. 1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HC1, 150 mM NaC1), High Salt

(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HC1, 500 mM NaC1), LiCl (0.25 M

LiC1, 1% Nonidet P-40, 1% Deoxycholate, 1 mM EDTA, 10 mM Tris-HC1), and TE (10 mM

Tris-HC1, 1.2 mM EDTA) wash buffers prior to the immune complexes being eluted from the

agarose beads with elution buffer (1% SDS, 0.1 M sodium bicarbonate). DNA (bound sample)

was de-crosslinked from histones with 10 Cl~/ml 5 M NaCl and then treated with 20mg/ml RNase

A and 40 Cpg/ml Proteinase K. DNA (bound and unbound fractions) was purified using a

QIAquick PCR Purification kit (Qiagen) before analysis by Taqman real-time PCR.

Taqman Real-Time PCR Analysis

Bound and unbound DNA was amplified by real-time PCR using TaqMan Universal PCR

Master Mix, No AmpErase UNG (Applied Biosystems) and FAM-labeled TaqMan target-

specific primer/probe. Reactions were run in triplicate in concentrations recommended by the

manufacturer. Primer and probe sequences are shown in Table A-1. PCR was performed and

analyzed using Applied Biosystems 7900HT Sequence Detection Systems. Cycle conditions

used were as follows: 500C for 2 min. (1 cycle), 950C for 10 min. (1 cycle), 950C for 15 sec., and

600C for 1 min. (40 cycles). Threshold values used for PCR analysis were set within the linear

range ofPCR target amplification. Average cycle threshold (Ct) values were determined, and

the relative quantity was calculated using a standard curve specific for the primer/probe set of

interest.









Results

The Chromatin Profile of the Latent HSV Genome in Rabbits Latently-Infected with Wild-
Type HSV-1 Is Similar to that Observed for Latently-Infected Mice

When the ChlP assay was performed on rabbit TG that were latently-infected with wild-

type 17syn+, the anti-dimethyl H3 K4 profie revealed an enrichment in the histone modification

for the LAT region relative to the lytic genes ICPO and ICP27 (Figure 2-2A). This was similar to

the findings obtained for the latently-infected mouse, in which transcriptional permissiveness of

the LAT 5' exon/enhancer was approximately 3.5 times greater than that of the LAT promoter

region and almost 40 times greater than that of the lytic genes, ICPO and ICP27 (Kubat et al.,

2004). Thus, it appears that wild-type HSV-1 establishes a similar latent chromatin profile in

both the footpad-infected mouse and the ocularly-infected rabbit.

Deleting the Core LAT Promoter Results in Increased H3 K4 Dimethylation of the LAT
Promoter Region in Latently-Infected Rabbits

The chromatin profile of HSV-1 genomes in latently-infected mice, in which the 5'

exon/enhancer is more transcriptionally permissive than the LAT promoter or nearby lytic genes,

is the same for both a wild-type and a promoter-deletion (LAT-negative) mutant (Kubat et al.,

2004). The profile observed in latently infected rabbits was strikingly different. As shown in

Figure 2-2B, on average the LAT promoter region (amplified by primers approximately 300 bp

upstream of the deletion) in the LAT core promoter deletion mutant, 17A~st, is 8.5 times more

enriched in dimethyl H3 K4 than the wild-type promoter region. The 5' exon/enhancer, ICPO,

and ICP27 all show similar levels of enrichment between the two viruses. Since the only region

with a dramatic difference between 17syn+ and 17A~st is the LAT promoter, this suggests that

the region deleted in 17APst contains a cis-element with repressive activity.









Neither 17syn+ nor 17APst Display Dynamic Changes in the LAT Region in Response to
Epinephrine Induction of Latently-Infected Rabbits

Since explant of mouse DRG appeared to induce dramatic changes in the chromatin profile

at early times post-explant, one would surmise that iontophoresis of rabbits would induce similar

changes. However, iontophoresis of rabbits at 0, 1, 2, and 4 h, resulted in no significant change

(Figure 2-3). As shown in Figure 2-4, the LAT promoter region of 17APst remains highly

enriched in dimethyl-H3 K4 relative to the other targets tested for all times examined. These

findings suggest that events related to chromatin remodeling might not occur as rapidly in the

rabbit as they do in the mouse or that during latent establishment in the rabbit, the LAT promoter

plays a different role than in the mouse in establishing a chromatin profile that is permissive for

normal reactivation to occur.

Relative H3 K4 Dimethylation Levels of the LAT Promoter Region in 17APst Are Higher
than Those of Wild-type for All Times Examined

The scale of bound/input (B/U) ratios for the 17APst ChlP experiment is much higher than

that observed for 17syn+, as visible by comparing Figures 2-3 and 2-4. The difference can be

appreciated by the comparison of the average values for the times examined. As shown in

Figure 2-5, when the ratios of the average 17A~st to 17syn+ dimethylation levels are determined,

the 5' exon/enhancer, ICPO and ICP27 all show ratios of approximately one, indicating similar

levels of dimethylation, while the LAT promoter ratios range from approximately 5 to 22-fold

more dimethylation in 17A~st than in 17syn+. This hyperdimethylation suggests that the region

deleted contains an element that normally represses H3 K4 dimethylation in the wild-type virus.

Discussion

Kinetics of Chromatin Remodeling

Upon explant of latently-infected murine DRG, a rapid loss of H3 K9, Kl4 acetylation of

the LAT 5' exon/enhancer is seen within the first hour of explant (Amelio et al., 2006). This









decrease is approximately 5-fold less than what is observed initially at 0 h.p.e. and precedes an

increase in acetylation of the ICPO promoter that occurs between 2 and 3 h.p.e. (Amelio et al.,

2006). This timing does not appear to be the same in the rabbit model. When the latent HSV-1

chromatin profile was assessed for rabbit TG infected with wild-type virus, the virus displayed a

similar latent profile to that of the latently-infected mouse. However, when the virus's chromatin

profile was examined after epinephrine-induced reactivation (1, 2, and 4 hours post-epinephrine

induction), it did not mirror the findings from the mouse explant experiments. In fact, little, if

any, change is observed at all within the first four hours of epinephrine-induction (Figure 2-3).

Since the timing of chromatin changes does not appear the same as is seen in the mouse, it

is possible that there are different mechanisms by which latent HSV-1 can reactivate. The mouse

DRG explant model is dependent on a more stressful reactivation stimulus, i.e., the removal of

tissue, while the rabbit eye model allows for a more clinically-relevant epinephrine-induced

reactivation. A more physiologically relevant stimulus might cause a more gradual change of

latent genome configuration. Perhaps at a single-cell level, changes occur quite rapidly, but they

do not translate to the level of the tissue until slightly later. For example, since the epinephrine is

administered to the eye and reactivation occurs in the ganglia, it might take some time for the

epinephrine to reach and stimulate all cells in the ganglia; in explant-induced reactivation, a

uniform stressor (ganglion removal from the animal) might stimulate cells to reactivate very

rapidly. Thus, it remains possible that if rabbit TG are analyzed for chromatin at later time

points, changes might be apparent, and these experiments are underway.

A Repressive Element in the LAT Promoter

When the chromatin profile of the LAT promoter deletion mutant, 17APst, is analyzed,

there is a striking increase in transcriptional permissiveness of the region slightly upstream of the

deleted region, while the remainder of the viral targets tested show levels of H3 K4









dimethylation that are comparable to wild-type (Figures 2-2). This suggests the presence of a

repressive element in the native core LAT promoter that might prevent increased transcriptional

permissiveness of the LAT region at inappropriate times, such as during lytic infection.

Additionally, since efficient reactivation from latency in the rabbit appears to require LAT

expression (Hill et al., 1990; Perng et al., 1994), LAT may play a role in establishing the genome

in a configuration compatible with reactivation. It has been proposed by Bloom et al. (1996) that

like XIST, a non-coding RNA that silences the inactive X chromosome, the LAT RNA acts in cis

to silence the HSV-1 genome during latency. Perhaps the LAT functions differently and is less

important in the mouse. Since explant of mouse DRG can induce reactivation of promoter-

deletion viruses that would not efficiently reactivate in the rabbit, a correct chromatin profile

may be less crucial for reactivation in the mouse.

The 202-bp core LAT promoter contains a number of binding sites for factors that may, in

turn, bind various chromatin modifiers. Further investigation of this region should yield valuable

insight into the mechanisms of HSV-1 reactivation.












R, U RL Rs Us Rs





...... .... ... .-- --- IC PO
Promoter Enhancer
I I

Mouse Wild-type


Mouse LAT-negative



Figure 2-1. Diagram of the chromatin profile of the latent HSV-1 genome in the mouse. The
LAT promoter is designated by the light gray box, while the LAT enhancer is shown
in dark gray. Green bars indicate regions of higher transcriptional permissiveness,
while red bars indicate less transcriptionally permissive regions.


U.015

0.01





O 25




0.2


0.15


I

~---.....~~~ ~ ~~~~~~~~~~~ ~~~~~~~~~


LAT


B p



1
B
B
;E!
I


Figure 2-2. LAT region histone H3 K4 dimethylation status of latently-infected rabbits for A)
wild-type 17syn+, or B) promoter-deletion mutant 17APst. Relative Bound/Unbound,
B/U, values are depicted for the LAT promoter, 5' exon/enhancer, ICPO, and ICP27.


+ LATPRo.
=5' Exon
AICPO
x ICP27










*IAT~Ro.
= 5'Exon
AICPO
x ICP27




























































































Figure 2-3. Dimethyl H3 K4 status during epinephrine-induced reactivation at A) 0, B) 1, C) 2,
and D) 4 hours post-epinephrine (h.p.e.) for wild-type 17syn+. Relative

Bound/Unbound, B/U, values are depicted for the LAT promoter, 5' exon/enhancer,

ICPO, and ICP27.


60






100

00





60











100





60


t
I


















r

1


A 10"


a80


e!60


+L.Pla
m5' emn
AICPO
ICP21














+ L Pro.
m9exon
A ICPO
ICP27














+L.PRo.


~ICIO















*L Pm.
n 5' exon
& ICPO
SICP27





H


-


-

-


A *"
00


33 *


TO ,

l00
100





B


430




2 00

100



8000
C
700

600
%: 00
dl400

i zoo3

100


80

D m



500



g 300


+L. P20.
5' exon
AICPO
= ICP27












+L Ro
= 5' emn
AICPO
SICP27












+L.PRo.
5' emn
AICPO
xlCP27












*L.Pro.
= 5' Exon
A ICRI
SICP217


Figure 2-4. Dimethyl H3 K4 status during epinephrine-induced reactivation at A) 0, B) 1, C) 2,
and D) 4 hours post-epinephrine (h.p.e.) for promoter-deletion mutant, 17APst.
Relative Bound/Unbound, B/U, values are depicted for the LAT promoter, 5'
exon/enhancer, ICPO, and ICP27.





















55'exan
10ICPO
5 ICP27





0 1 2 4
Time (hp.L)



Figure 2-5. Ratios of average relative H3 K4 dimethylation of 17APst to those of 17syn+ for
epinephrine-induction in rabbits (0, 1, 2, and 4 hours post-induction). Ratios for the
LAT promoter (L. Pro.), LAT enhancer (5' exon), ICPO, and ICP27 are shown.









CHAPTER 3
INVESTIGATION OF THE ROLE OF A LAT PROMOTER cAMP RESPONSE ELEMENT
(CRE) IN REACTIVATION

Objective

It has been previously shown that LAT abundance transiently decreases following explant-

induced reactivation, suggesting that regulation of LAT expression may be an important

component of the switch between latent and productive infection. The LAT promoter contains

various elements that may be important regulators of the LAT region during latency and

reactivation. The studies described here were performed to investigate the role of one of the

LAT promoter's cAMP response elements (CREs) in reactivation. To do so, a virus was

constructed with a site-directed mutation in the CRE and assessed for (1) in vitro replication in

cell culture, (2) replication and virulence in the mouse, (3) ability to establish latency and

express the LAT, (4) chromatin profile during latency, and (5) ability to reactivate from latency

in the rabbit.

Background and Previous Findings

Since reactivation of HSV-1 from latency is a stress-inducible phenomenon, it seems

plausible that the stress-responsive cAMP pathway could play a role in regulation, particularly

since the LAT promoter contains two CREs (Leib et al., 1991; Kenny et al., 1994). The cAMP

pathway's cascade of events is triggered upon epinephrine binding to P-adrenergic receptors of

cells. Once binding occurs, adenylyl cyclase converts ATP to cAMP (Tao and Lipmann, 1969),

which can then activate the catalytic subunit of protein kinase A (PKA). This subunit

translocates to the cell nucleus, where it can phosphorylate members of the cAMP response

element binding (CREB) protein family (Montminy and Bilezikjian, 1987; Yamamoto et al.,

1988). The CREB activator proteins contain a kinase-inducible domain (KID) flanked by

glutamine-rich regions at the amino-terminus and a basic region/leucine zipper (bZIP) domain at









the carboxy-terminus, which is important in DNA binding and nuclear translocation (Dwarki et

al., 1990; Waeber and Habener, 1991; Xing and Quinn, 1994). Since the CREB gene undergoes

alternative splicing to yield various family members, truncated versions of the protein that lack

an activation domain, can cause transcriptional repression rather than activation (Karpinski et al.,

1992; Molina et al., 1993).

Once CREB is phosphorylated and activated at a gene's promoter, it facilitates recruitment

of CREB-binding protein (CBP) (Chrivia et al., 1993), which in turn can promote transcriptional

activation (Arias et al., 1994; Kwok et al., 1994). Although some questions remain as to the

exact role of CBP in transcriptional activation, it has been implicated in promoting rapid

formation of the preinitiation complex (PIC) to increase the rate of transcription and has also

been suggested to facilitate recruitment of mediator complexes to active sites of transcription

(reviewed in Vo and Goodman, 2001). Because CBP is a histone acetyltransferase (HAT), it

likely contributes to promoter activation through chromatin remodeling (Ogryzko et al., 1996).

Genome-wide characterization of CREB binding to various target genes in human tissues

revealed that while CREB occupies approximately 4,000 promoters, only a small subset of those

genes are actually activated in response to cAMP, which is likely due to a requirement for

coactivator recruitment (Zhang et al., 2005). In other words, CREB binding is constitutive, and

regulation is dependent on CBP and possibly, other coactivators. In contrast, one study

demonstrated, through the use of chromatin immunoprecipitation (ChIP) assays, that binding is

regulated in a cell-specific manner, correlating with the potential for gene expression (Cha-

Molstad et al., 2004). This study also found that histone H13 K4 dimethylation corresponds to

CREB binding, suggesting either that CREB binding to a promoter is regulated in an epigenetic









manner or that CREB binding is indicative of transcriptional permissiveness (Cha-Molstad et al.,

2004).

It was previously determined that the HSV-1 LAT promoter contains a functional cAMP

response element (CRE) with complete homology to the proenkephalin CRE (Leib et al., 1991).

This element, referred to herein as the -43CRE due to its starting location relative to the LAT

transcriptional start site, was shown to be inducible upon application of known modulators of

intracellular cAMP levels, and it was also shown to bind the wild-type form of the CRE binding

(CREB) protein in vitro (Leib et al., 1991; Millhouse et al., 1998). Interestingly, when mobility-

shift assays were performed using PC-12 cell nuclear extracts, results suggested that a protein or

proteins other than CREB can bind specifically to the LAT -43CRE (Leib et al., 1991); however,

this observation was not further explored.

The role of the -43CRE in reactivation was also examined in vivo using a recombinant

virus, in which the mutated binding site was predicted to provide less than 10% CREB binding

(Bloom et al., 1997). When the recombinant virus was tested in the rabbit eye model,

epinephrine induction yielded reactivation that was intermediate (58%) between wild-type strain

17syn+ (78%) and promoter-deletion mutant 17APst (19%) (Bloom et al., 1997). Spontaneous

reactivation levels were similar between the -43CRE mutant virus and 17A~st (19% and 16%,

respectively) (Bloom et al., 1997). In the rabbit studies, as well as in studies performed using the

mouse hyperthermic stress model, the mutation in the -43CRE mutant virus did not appear to

affect establishment or maintenance of latency (Bloom et al., 1997; Marquart et al., 2001). Thus,

the -43CRE seems to have a role in reactivation. Because epinephrine can stimulate P-

adrenergic receptors to begin the cAMP cascade, it seems plausible that the induction of

reactivation is tied to the responsiveness of the -43CRE. In fact, when the P-adrenergic receptor-









blocker, propanolol, was inj ected into mice latently infected with HSV-1, the appearance of

infective virus was decreased significantly in the tear film, cornea, and trigeminal ganglia

(Gebhardt and Kaufman, 1995), and when it was used to treat latently-infected rabbits,

spontaneous reactivation was reduced (Kaufman et al., 1996). Therefore, the cAMP response

pathway may play a role in HSV-1 reactivation from latency.

Approximately 40 base pairs upstream of the -43CRE exists a second CRE, which will be

referred to as the -83CRE. This element was identified through chloramphenicol

acetyltransferase (CAT) activity assays of various promoter deletion mutants and was suggested

to convey cell-specifieity, as determined through testing in C1300 mouse neuroblastoma cells

and L929 mouse fibroblast cells (Kenny et al., 1994). Specifically, when promoter sequences

including the -83CRE were added to the neuronal cells in plasmid-based transient expression

assays, a three- to fourfold increase in promoter activity was observed, while no effect was seen

in nonneuronal cells (Kenny et al., 1994). Upon further examination of the -83CRE, it was

determined through electrophoretic mobility shift assays (EMSA) using C1300 cell nuclear

extracts that although the site can bind both CREB-1 and CREB-2, it binds CREB-2 with much

higher affinity (Millhouse et al., 1998). CREB-2, when over-expressed in vitro, causes a

significant repression of CRE-mediated transcription due to a lack of phosphorylation sites

(Karpinski et al., 1992). Thus, in the HSV-1 LAT promoter, it is possible that the site plays a

role in the repression of LAT transcription that is observed during early times post-explant

(Amelio et al., 2006).

One might envision the LAT promoter as a molecular switch that controls LAT expression

to regulate the transition between latency and reactivation, especially since transcription of ICPO,

an IE gene, occurs downstream of the LAT promoter and in an antisense orientation to the LAT,









with the potential for overlap of both transcripts. The ICPO protein has several defined

functions, including transactivation of various cellular and viral genes, host protein degradation,

and viral localization to ND10 cellular structures (reviewed in Everett, 2000). ICPO mutants are

viable and can establish latent infection, although they display reduced replication in cell culture

at low m.o.i. (Stow and Stow, 1986; Sacks and Schaffer, 1987; Everett, 1989) and inefficient

reactivation from latency (Leib et al., 1989; Cai et al., 1993). Interestingly, the inefficient

growth by the ICPO mutants can be restored to near wild-type levels when grown at high m.o.i.

in cell culture (Sacks and Schaffer, 1987). The wild-type ICPO promoter can be activated in

response to stress, as demonstrated through stress-stimulating experiments in ICPO reporter

transgenic mice (Loiacono et al., 2003). Additionally, the chromatin surrounding the ICPO

promoter becomes more transcriptionally permissive within four hours of murine DRG explant-

induced reactivation (Amelio et al., 2006). While it appears that there may be some role for

ICPO in reactivation, it is not clear whether ICPO plays a critical role in facilitating IE

transcription at very early times during reactivation, or whether it functions as a general

transactivator of transcription that enhances the reactivation process.

Because of the potential importance of the -83 CRE in regulating the LAT promoter and

possibly in facilitating reactivation the experiments described here were designed to

characterize and define the function of the HSV-1 LAT promoter -83CRE site. Creation of a

recombinant virus with an eight base-pair mutation in the -83 CRE site of the LAT promoter

allowed the relevance of the site in the acute, latent, and reactivation phases of the viral lifecycle

to be examined.









Materials and Methods

Plasmid Generation, Mutagenesis, and Purification

Plasmid pNG1 was generated through the subcloning of the 1.2 kilobase fragment of the

LAT promoter through the 5' exon contained between the Dral and BstEII restriction enzyme

sites (nt 118,002-119,202) into pBluescript II.

The resulting plasmid was subj ected to site-directed mutagenesis of the -83CRE site

(AATTACA) to a BglII restriction enzyme site using Stratagene's Quikchange II Site-Directed

Mutagenesis kit. Nucleotides were mutated in groups of four using the following sets of primers:

P1Sense--GCA GAC GAG GAA AAT AAA ACA GAA TCA CCT ACC CAC GTG GTG

CTG TGG; PlAntisense--CCA CAG CAC CAC GTG GGT AGG TGA TTC TGT TTT ATT

TTCCTC GTC TGC; P2Sense-GCA GAC GAG GAA AAT AAA ACA GAT CTT CCT ACC

CAC GTG GTG CTG TGG; P2Antisense--CCA CAG CAC CAC GTG GGT AGG AAG ATC

TGT TTT ATT TTC CTC GTC TGC. The mutagenesis reaction was performed according to

manufacturer' s instructions using 50 ng of starting plasmid, and thermal cycler conditions were

as follows: 1 cycle of 30 sec. at 950C and 16 cycles of 30 sec. at 950C, 1 min. at 550C, and 4

min. at 680C. Because the mutated site created a BglII restriction enzyme site not present in the

parental DNA, mutated plasmid DNA was subj ected to restriction endonuclease digestion with

that enzyme to further verify that the correct mutation was obtained. Additionally, plasmids

were sequenced for verification of the correct mutation.

Upon confirmation of the desired mutation, the plasmid DNA was grown in E. coli cells

and purified using a cesium chloride gradient (Garger et al., 1983). Briefly, the plasmid was

grown in 2x YT media containing ampicillin, pelleted, and resuspended in glucose buffer (50

mM glucose, 25 mM Tris-HC1, pH 8.0, 10 mM Na2EDTA). Bacterial cells were lysed in 20

mg/ml lysozyme and lysis solution (0.2 N NaOH, 3% SDS, and water to volume). Potassium









acetate solution (5 M potassium acetate and 11.5% glacial acetic acid) was added and incubated

in an ice water bath for 15 min. The sample was centrifuged, the supernatant was filtered and

chloroform:isoamyl alcohol (24:1) extracted, and the plasmid precipitated with isopropanol. The

pellet was resuspended in lx TE (10 mM Tris-HC1, pH 8.0, 1 mM EDTA), combined with 4 g

cesium chloride, and 4 mg ethidium bromide. After overnight centrifugation at 44,000 rpm

(176,284 x g) in a VTi 65.2 vertical rotor, the plasmid DNA band, visible upon exposure to

ultraviolet light, was removed using a syringe and extracted against isoamyl alcohol to remove

the ethidium bromide. DNA was precipitated, phenol:chloroform extracted, precipitated again

and resuspended in lx TE.

Cells and Viruses

L7 cells, a Vero cell line containing ICPO stably transfected (Samaniego et al., 1997),

were a gift from the lab of Neal DeLuca. Rabbit skin (RS) and L7 cells were grown at 370C in

the presence of 5% CO2 in minimal essential media (MEM) supplemented with 5% calf serum

(RS cells) or 10% fetal bovine serum (L7 cells), 292 Cpg/ml L-glutamine, and antibiotics (250

U/ml penicillin and 250 Cpg/ml streptomycin). Neuro-2A (C1300) cells were obtained from the

American Type Culture Collection and grown at 370C in the presence of 5% CO2 in MEM

supplemented with 10% fetal bovine serum, 292 Cpg/ml L-glutamine, antibiotics (250 U/ml

penicillin and 250 Cpg/ml streptomycin), and 1 x Non-Essential Amino Acids (Mediatech, Inc.).

For transfections, RS cells were subj ect to overnight serum-starvation at 3 1.50C (5% CO2) with

MEM supplemented with 1.5% fetal bovine serum, in addition to L-glutamine and antibiotics as

described above.

HSV-1 strain 17syn+ was obtained as a low passage stock from J. Stevens. 17A~st-

Stuffer (replacement of nucleotides 1 18,666 to 1 18,869 of 17syn+ with the Kpnl-SacI fragment










of pBluescript' s multiple cloning site [MCS]) was constructed by D. Bloom. -83CRE and F8-1

were constructed as described below.

DNA Isolation for Transfections

17APst-Stuffer virus was used as the backbone for the construction of the -83 CRE viral

recombinant containing the site-directed mutation of the -83CRE. The -83CRE recombinant was

used in the construction of the rescuant, F8-1. In order to prepare the viral DNA used for

transfections, virus was cultivated on RS cells at an m.o.i. of 0.01. Upon appearance of

cytopathic effect (CPE), infected cells were harvested, centrifuged, and the pelleted cells

resuspended in hypotonic lysis buffer (10 mM Tris, pH 8.0, 10 mM EDTA, 0.5% Nonidet P-40,

0.25% NaDOC). After 5 min. incubation on ice, the sample was centrifuged, the supernatant

removed and incubated with 1 mg/ml Proteinase K and 1% SDS for 1 h at 50.C; 1 mg/ml

Proteinase K was added after the initial incubation and incubated for 1 h more at 50.C. The

sample was extracted with phenol, chloroform, and isoamyl alcohol. Viral DNA was

precipitated by the addition of 0. 1 vol 3 M NaOAc, followed by the very slow addition of 2 vol

ice cold 100% ethanol. The DNA was spooled, removed, air-dried and resuspended in lx TE.

Virus Construction and Plaque Purification

Plasmid DNA was transfected with viral DNA to allow for recombination. The mutated

-83 CRE plasmid DNA was first digested using BspEl and BsaBI and was then gel purified.

Approximately 4 Cpg of the purified, linearized plasmid DNA was combined with varying

amounts of 17APst-stuffer DNA (2 Cpg, 4 Cpg, 8 Cpg, and 16 Cpg) in a final volume of 225 Cl1 TNE

buffer (10 mM Tris, pH 7.4, 1 mM EDTA, and 0.1 M NaC1) plus 25 Cl~ CaCl2. Rescuant F8-1

was constructed by combining -83CRE viral DNA with plasmid pAatlI (nt 4817-9271 of

17syn+). The DNA was precipitated by the addition of 2 x HEPES while blowing bubbles

through a pipet and allowed to incubate for 20 minutes at room temperature. This transfection









mix was then applied to RS cells which had undergone an overnight serum-starvation (described

above), and this was allowed to incubate at room temperature for 30 min. After this incubation,

MEM supplemented with 1.5% fetal bovine serum was added to the cells and incubated for 4

hours at 370C in the presence of 5% CO2. This incubation was followed by the addition of

hypotonic lysis buffer (10 mM Tris, pH 8.0, 10 mM EDTA, 0.5% Nonidet P-40, 0.25% sodium

deoxycholate) directly to the monolayer, followed by three washes with media. Supplemented

MEM containing 5% calf serum was added to the cells, which were then incubated at 370C in the

presence of 5% CO2 for 3-4 days (when CPE was observed).

Screening for recombinant viruses was performed by picking plaques followed by dot

blot hybridization. The virus resulting from the transfection was diluted to yield well-separated

plaques (10-4 through 10-'), and 0.5 ml of these dilutions plated onto confluent monolayers of

cells in 60-mm tissue culture dishes (two to three dishes per dilution). After 1 hour of

adsorption, monolayers were overlayed with 0.75% agarose in lx supplemented media, and the

dishes were then incubated until plaques were visible (2-3 days). To assist in visualization of

plaques, dishes were counterstained with a 1:30 dilution of 3.3 g/L Neutral Red (Sigma) in

unsupplemented media for approximately 4-6 hours. The liquid overlay was removed, and

plaques were picked using sterile Pasteur pipettes. After gently aspirating into the pipette, the

plaques were expelled into a 96-well dish containing 150 Cl1 media per well. Once plaques were

picked, the dish was frozen at -800C and then thawed in the tissue culture incubator. 50 Cl1 of the

plaque suspensions were inoculated onto confluent cells in a 96-well dish and allowed to adsorb

for 1 hour. At the end of the incubation, supplemented media was added to each well, and the

dishes were incubated until 100% CPE was observed (approximately 3 days). A Millipore dot-

blot apparatus was used to transfer 50 Cl~ of infected cells onto a nylon membrane, which was









washed with 200 Cl1 of a solution containing 1.5 M NaCl and 0. 1 M NaOH, followed by 200 Cl~ of

a solution containing 0.2 M Tris, pH 7.5, and 200 Cl~ of a solution containing NaC1, NaH2PO4,

and EDTA. Once the solutions were through the apparatus, the membrane was baked for one

hour at 800C.

Dot blots were probed with radioactively labeled random-primed DNA probes prepared

using the Pst-Pst fragment of the HSV-1 LAT promoter, the MCS fragment of pBluescript, or a

22-mer encompassing the wild-type -83CRE of the LAT promoter. Probes were labeled with [a-

32P]dCTP using the Rediprime II Random Prime Labelling System (Amersham Biosciences),

according to the manufacturer' s protocol.

To screen for the recombinant -83CRE virus, the membrane was prehybridized for 4-5

hours at 62.50C with a solution containing 3 M NaC1, 0.3 M sodium citrate, 50 mg/ml nonfat dry

milk and 2 Cpl/ml Antifoam A, the labeled probe added to the prehybridization solution and the

dot blot hybridized overnight. The membrane was then washed at room temperature once for 5

min. with a wash solution containing 0.3 M NaC1, 0.06 M Tris-HC1, and 0.002 M EDTA and

twice for 5 min. each with a wash solution containing 0.03 M NaC1, 0.006 M Tris-HC1, and

0.0002 M EDTA. The blot was dried and exposed to X-ray film overnight.

To screen for the rescuant virus, F8-1, tetramethyl ammonium chloride (TMAC)

hybridization was used. The membrane was prehybridized for 1 hour at 580C with a

hybridization solution consisting of 3 M TMAC, 0. 1 M NaPO4, pH 6.8, 1 mM EDTA, pH 8.0, 5

x Denhardt's Solution, 0.6% SDS, and 100 Clg/ml denatured Salmon Sperm DNA. For the

hybridization, the hybridization solution was replaced with fresh solution, and a labeled 22-mer

probe identical to the wild-type promoter region encompassing the -83CRE site was hybridized

to the membrane for 24 to 48 hours at 580C. The membrane was washed twice at room









temperature with Wash #1 (3 M TMAC, 50 mM Tris, pH 8.0, 0.2% SDS), once with Wash #1 at

600C for one hour, and twice at room temperature with Wash #2 (2 x SSPE, 0.1% SDS, 1 mM

EDTA, pH 8.0). The membrane was then dried and exposed to X-ray film. F8-1 was one of two

independent plaques identified from the initial screen and purified through 4 rounds of plaque

purification.

PCR Analysis

The LAT promoter regions of the recombinants were analyzed by PCR performed on

viral DNA that was isolated as described above (see Cells and Virus Cultivation/DNA Isolation

for Transfections). Amplification reactions contained lx GoTaq" Green Master Mix (Promega),

600 ng each of primer Upfragment, 5'-CGA GGA ACA ACC GAG GGG AAC (nt 1 18,305-

118,325) and Downfragment, 5'-CTG AGA TGA ACA CTC GGG GTT ACC (nt 119, 179-

119,202), 50 ng of viral DNA and nuclease-free water to a final volume of 50 pl~. The DNA was

amplified using an Ericomp thermal cycler (San Diego, CA) with 2 min. at 950C (one cycle), 3

min. each at 940C, 550C, and 720C (one cycle), and 1 min. each at 940C, 550C, and 720C (three

cycles). PCR products were visualized on 1% agarose gel containing ethidium bromide.

Growth Curves

In order to assess replication of the -83CRE recombinant in various cell lines, the virus

was inoculated at a multiplicity of infection (m.o.i.) of 0.001, 0.01, or 5. The virus, along with

its rescuant (F8-1) and the wild-type 17syn+ virus, was used to infect confluent RS cells, L7

cells, or Neuro-2A cells grown in 35-mm cell culture dishes. Time (hours post-infection) was

monitored starting one hour after the inoculum was added to the cells. Infected cells were

harvested by gentle scraping of the dishes at 0, 8, 24, 48, 72, and 96 h.p.i. for multi-step growth

curves (low m.o.i.) or 0, 8, 24, and 48 h.p.i. for single-step growth curves (high m.o.i.). After

one freeze-thaw, viral titers were determined.









Mouse Survival Assay

Six to 8 week old female ND4 Swiss mice (Harlan Sprague Dawley, Inc.) were infected

via the footpad with 500 pfu, 5,000 pfu, or 50,000 pfu. Briefly, mice were injected with saline in

the rear footpads to soften the keratinized epithelium. Three to four hours later, mice were

anesthetized intramuscularly with 0.01 to 0.02 ml of a cocktail of ketamine (30 to 45 mg/kg),

xylazine (7.5 to 11.5 mg/kg) and acepromazine (2.5 to 3.75 mg/kg). During this time, the

footpads were abraded with an emery board and virus was applied. Viral absorption occurred

during the time that the mice remained under anesthesia (approximately 45-60 minutes). The

number of mice surviving throughout the acute phase of infection was assessed.

Intracranial Inoculation as an Assay for Neurovirulence

In order to assess the neurovirulence of the recombinant virus, ND4 Swiss mice were

anesthetized using isofluorane and inoculated intracranially with 10 Cll of dilutions of virus using

a 27 V/2 gauge needle. Doses given were 10 pfu and 100 pfu. Mice were monitored for survival

over the acute phase of infection.

DNA Extraction and Analysis of Course of Infection

Mice were infected via the footpads with 5,000 pfu/mouse (8 hour sample) or 10,000

pfu/mouse (2 and 4 day samples) of either 17syn+ or -83CRE virus. At 8 hours, 2 days, or 4

days post-infection, 4-5 mice from each group were sacrificed, and their feet, DRG, and spinal

cords were snap-frozen in liquid nitrogen. Tissue samples were homogenized in 4 ml (feet) or

0.2 ml (DRG and spinal cord) TES (10 mM Tris, pH 7.4, 0.1 M NaC1, 1 mM EDTA) with Duall

glass dounces and treated with 1% SDS and 1 mg/ml Proteinase K overnight at 500C. Samples

were phenol :chloroform:isoamyl alcohol extracted, chloroform:isoamyl alcohol extracted, and

ethanol precipitated. After air-drying, the DNA pellet was resuspended in 1xTE. Spinal cord

and DRG samples were diluted 1:10 and foot samples were diluted 1:100 for use in real-time









PCR. HSV-1 genome equivalents were determined through calculation of the relative quantity

of PCR amplification of the HSV-1 polymerase gene normalized to the PCR amplification

products of the cellular gene target, XIST.

RNA Isolation and Reverse Transcription for Acute RNA Levels in Cell Culture

RS or Neuro-2A cells were seeded for next day confluency in 35-mm tissue culture

dishes. Upon confluency cells were infected at an m.o.i. of 0.01 with either 17syn+ or -83CRE

virus. One ml of viral inoculum in MEM was applied to each dish, allowed to adsorb into the

cells for one hour, and then replaced with fresh supplemented MEM. After this point, cells were

harvested for RNA at 2, 4, 6, or 8 hours post-infection (with timing started after the application

of fresh media) using 1 ml Trizol, which was triturated to lyse cells. Samples were transferred to

Eppendorf tubes, incubated at RT for 5 min., and 0.2 ml chloroform was added to each tube,

vortexed and incubated at RT for 2 min. Tubes were centrifuged at 12,000xg for 15 min. at 40C

before the aqueous phase was transferred to a new tube and the RNA was precipitated by the

addition of 0.5 ml isopropanol. Samples were incubated at RT for 10 min. and centrifuged at

12,000xg for 10 min. at 40C. After the removal of the supernatant, the RNA pellet was washed

with 1 ml 70% EtOH and spun at 7,500xg for 5 min. at 40C. This supernatant was removed and

the pellet was air-dried briefly before being resuspended in 45 Cl1 nuclease-free H20.

Upon resuspension of the precipitated RNA, DNase treatment was performed using Turbo

DNA-free (Ambion) according to the manufacturer' s instruction. Reverse transcriptions were

performed with Omniscript reverse transcriptase (Qiagen) in reaction volumes of 20 ul. Briefly,

reactions contained Omniscript lx buffer, 0.5 mM each dNTP, 1 CIM random decamer primer

(Ambion), 10 units/ul SUPERase-In (Ambion), 1 Clg RNA, 8 units Omniscript reverse

transcriptase, and RNase-free water to volume. Additionally, RNA controls ("No RT" controls)









were performed using the same concentration of RNA in water. Reactions were incubated at

370C for one hour.

Taqman Real-time PCR Analysis

cDNA or DNA was amplified by real-time PCR using TaqMan Universal PCR Master

Mix, No AmpErase UNG (Applied Biosystems) and FAM-labeled TaqMan target-specific

primers/probes (Applied Biosystems, Inc.) (Table A-1). Reactions were run in triplicate in

concentrations recommended by the manufacturer. Primer and probe sequences are listed in

Appendix A. PCR was performed and analyzed using Applied Biosystems 7900HT Sequence

Detection Systems. Cycle conditions used were as follows: 500C for 2 min. (1 cycle), 950C for

10 min. (1 cycle), 950C for 15 sec., and 600C for 1 min. (40 cycles). Threshold values used for

PCR analysis were set within the linear range of PCR target amplification. Average cycle

threshold (Ct) values determined in triplicate were averaged, and the relative quantity was

calculated using a standard curve specific for the primer/probe set of interest. Briefly, Ct values

for 10-fold dilutions of DNA (viral or cellular) of known concentration were determined and

graphed as a function of dilution. The equation of the resulting line was used to extrapolate the

relative quantity of the sample of unknown concentration.

RNA Isolation and Reverse Transcription for Explant Studies

Mice (infected as described above with 400-500 pfu of virus) were euthanized by an

overdose of isoflourane, followed by cervical dislocation. Dorsal root ganglia (DRG) from

groups of two mice (8 ganglia per mouse) were removed as quickly as possible (3 to 5 minutes

per mouse) and placed in 0.5 ml RNA Later (Ambion). RNA was isolated from the tissue using

the method of Chirgwin, et al. (1979). Briefly, DRG were homogenized in guanidine

thiocyanate solution and 100 Cll removed for DNA isolation. The homogenate was layered on a

5.7 M cesium chloride cushion and centrifuged overnight at 30,000 rpm (111,132 x g) in a SW









41 Ti rotor. The supernatant was aspirated from the RNA pellet, which was briefly dried and

then resuspended in nuclease-free water. RNA was precipitated overnight using 0.1 vol 3 M

sodium acetate and 2 vol 100% ethanol. The DNA fraction of the sample was isolated as

described by Kramer and Coen (1995). Briefly, the DNA was precipitated overnight with 0.1 vol

3 M sodium acetate and 2 vol 100% ethanol. The resulting pellet was resuspended in 50 Cl1 of

DNA resuspension solution containing: 0.2 Cpg/ml proteinase K, 0.02% Tween 20, 1 x PCR

buffer (50 mM KC1, 10 mM Tris-HC1, pH 9.0, 0. 1% Triton X-100), and 1.5 mM MgCl2. The

DNA was incubated at 650C for 2 h, 800C for 20 min., and 940C for 10 min. (to inactivate

enzyme). Because of the presence of precipitate, the DNA sample was diluted 1:10 prior to use

in real-time PCR analysis.

Upon resuspension of the precipitated RNA, DNase treatment was performed using

Turbo DNA-free (Ambion) according to the manufacturer's instruction. Reverse transcriptions

were performed with Omniscript reverse transcriptase (Qiagen) as described above (see RNA

Isolation and Reverse Transcription for Acute RNA Levels in Cell Culture).

ChlP Analysis

Dorsal root ganglia were removed and pooled from 3 mice per time point. After

incubating in media at 370C for a given amount of time (0, 0.5, 1, 2, or 4 h post-explant), ganglia

were homogenized in 0.5 ml phosphate-buffered saline (PBS) in the presence of protease

inhibitors (1 Cpg/ml aprotinin, 1 Cpg/ul leupeptin, and 1 mM PMSF). DNA-histone complexes

were cross-linked by the addition of 37% formaldehyde to a final concentration of 1%. After the

addition of 0. 128 M glycine, the sample was pelleted and washed three times with PBS

containing protease inhibitors as described above. Samples were pelleted, resuspended in SDS-

lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HC1), and sonicated (Fisher Sonic

Dismembrator 100) to yield fragments of 500-1000 bp (setting 4, 2 bursts of 40 sec. each









followed by 1 burst of 20 sec.). Sonicated samples were pre-cleared with Salmon Sperm

DNA/Protein A Agarose beads (Upstate), and histone-DNA complexes were immunoprecipitated

overnight with 3.5 Cpg/ml of anti-acetyl-Histone H3 (Upstate). Prior to the wash steps, 25% of

the sample was removed and retained as the "unbound" fraction. Complexes were washed with

Low Salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HC1, 150 mM NaC1), High

Salt (0. 1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HC1, 500 mM NaC1), LiCl (0.25

M LiC1, 1% Nonidet P-40, 1% Deoxycholate, 1 mM EDTA, 10 mM Tris-HC1), and TE (10 mM

Tris-HC1, 1.2 mM EDTA) wash buffers prior to the immune complexes being eluted from the

agarose beads with elution buffer (1% SDS, 0.1 M sodium bicarbonate). DNA (bound sample)

was de-crosslinked from histones with 10 Cl~/ml 5 M NaCl and then treated with 20mg/ml RNase

A and 40 Cpg/ml Proteinase K. DNA (bound and unbound fractions) was purified using a

QIAquick PCR Purification kit (Qiagen) before analysis by Taqman real-time PCR.

Rabbit Reactivation

One to 2 kg New Zealand White rabbits were infected and housed at the Louisiana State

University Health Science Center's Animal Facility. Each rabbit eye received topical

proparacaine-HCI anesthetic prior to corneal scarification. Rabbits were infected with either

17syn+ or -83CRE virus inoculum at 200,000 pfu/eye. At days 3, 5, and 7 post-infection (p.i.),

the infections of the rabbit eyes were monitored by slit lamp examination for the presence of

dendrites on the corneas. Clinical scores (1 to 5, with 5 being the most severe) were assigned to

reflect the relative surface area of the cornea covered with dendrites as a means of assessing the

severity of the infection.

After 28 days p.i., rabbits were anesthetized with ketamine/xylazine and a solution of

0.015% epinephrine in sterile water (dissolved by the addition of one drop of HC1) was

iontophoresed into the rabbit corneas by applying current for 8 minutes at 0.8 mAmps. At










specified times post-iontophoresis, rabbits were euthanized with sodium pentobarbital and

decapitated for removal of TG. The TG were removed as rapidly as possible (5 to 15 min per

rabbit).

Results

The -83CRE virus construction was not straightforward. After a mutation was made to

the -83CRE site in a plasmid containing the LAT promoter and verified through sequencing, the

mutant plasmid was allowed to homologously recombine with DNA from virus 17APst-Stuffer, a

LAT-negative mutant containing a bacterial stuffer in place of the core LAT promoter. To

ensure that the recombinant used for characterization experiments was not made up of a mixed

population of viral genomes (e.g., the parental stuffer and the nascent recombinant), the virus

was plaque-purified, which permits selection for purity. Although the plaque purification

appeared to produce a pure recombinant virus after approximately seven rounds of purification

based on the dot-blot hybridizations, when the promoter region was amplified through PCR

(Figure 3-1), two products appeared for the supposed "pure" recombinant (Figure 3-2A). The

product of higher molecular weight corresponded to the recombinant, while the lower molecular

weight product was representative of the parental virus, 17APst-Stuffer. This suggested that the

virus was persisting as a mixed population of recombinants and wild-type virus or more likely as

a "single-sided" recombinant, in which only one of the two long repeats (RL) COntained the LAT

promoter -83 CRE mutation (see Figure 3-2). Since the lab had never had a recombinant that

needed more than 4 rounds of plaque-purification to achieve purity and because the PCR data

was consistent with a single-sided virus, this suggested that there might be a bias against making

the -83CRE mutation in both copies of the LAT. Because the -83CRE mutation could

potentially disrupt regulation of LAT expression and affect ICPO in the process, the virus was

plaque-purified on L7 cells, a Vero-cell derived cell line, in which ICPO is stably expressed









(Samaniego et al., 1997). Since the parental 17A~st-Stuffer virus used in the transfection

contained a pBluescript multiple cloning site (MCS) stuffer, the MCS fragment was used as a

negative hybridization control. After four rounds of plaque purification on L7 cells, dot blot for

the MCS stuffer indicated that the region did not exist in the -83CRE virus (not shown), and the

PCR product corresponding to the parental virus was no longer visible, suggesting that

complementing ICPO with L7 cells allowed a virus with the mutated -83CRE in both copies of

the LAT promoter to be purified to homogeneity (Figure 3-2B). As will be discussed further

later, it was determined upon characterization of the -83 CRE virus that a second site mutation

also occurred somewhere in the viral genome.

The -83CRE Recombinant's Replication Is Altered during the Lytic Phase of the Infection

After the -83CRE recombinant virus was purified and the correct mutation in the LAT

promoter confirmed through PCR analysis and sequencing, it was tested for replication in cell

culture. Wild-type 17syn+ and -83CRE virus were used to infect RS cells, L7 cells, and Neuro-

2A cells at the low m.o.i. of 0.01 to allow for multiple rounds of replication. As shown in Figure

3-3A, the -83CRE displayed approximately 10-fold more efficient replication than 17syn+ during

the first 8-24 hours of infection when assayed on RS cells, which are epithelial in origin. The

opposite was observed for the -83CRE' s growth on Neuro-2A cells, which produced yields of

approximately 10-fold less than 17syn+ throughout the times tested (Figure 3-3B). Since the

mutant was purified on L7 cells, it was tested for growth on that cell line as well. Providing

excess ICPO had no maj or effect on the -83CRE mutant' s replication, since relative yields on L7

cells were the same as on RS cells, with the mutant again replicating slightly more efficiently

than the wild-type virus early in the infection (Figure 3-3C).

Since the recombinant was not able to be purified until it was plagued on L7 cells, an

ICPO-complementing cell line, the possibility existed that ICPO function was altered in the









-83CRE. ICPO-deleted viruses show a multiplicity-dependent effect on replication, in which the

mutant grows very inefficiently at low m.o.i. but near wild-type levels of replication are restored

at high m.o.i. (Sacks and Schaffer, 1987). To determine whether the -83CRE mutation had a

multiplicity-dependent effect on replicative yields, single-step growth curves (m.o.i. of 5) were

performed using RS and Neuro-2A cells. As shown in Figure 3-4, the -83CRE recombinant

shows a similar effect to that observed for low m.o.i. on RS cells but the decreased replication on

Neuro-2A cells that was seen for the low m.o.i. infection was not observed. In fact, the -83CRE

demonstrated slightly enhanced replication (approximately 5-fold) relative to wild-type at 12

hours p.i. when grown on Neuro-2A cells. Thus, these data suggest a multiplicity-dependent

effect of the -83CRE recombinant on viral growth on the Neuro-2A cell line, but not on the RS

epithelial cells.

The -83CRE Recombinant Displays Impaired Replication and Spread in the Nervous
System of the Mouse

Because the -83CRE mutant virus showed some variation from wild-type in replication

assays, it was important to test the virus in the mouse to determine what biological effects the

mutation would have. Ten ND4 Swiss mice per dose per virus were infected with 500, 5000, or

50000 pfu of either 17syn+ or the -83CRE virus and were monitored closely during the acute

phase of infection (first three weeks of infection). The survival of mice at the varying doses is

shown in Figure 3-5 and in Table 3-1. While 17syn+ resulted in 0-30% survival for the doses

tested, mice infected with the -83CRE virus displayed a 90% survival rate for all doses,

indicating an LD5o of >5x104 pfu. Because of this dramatic deficit in virulence following

infection via the mouse footpad, we sought to determine whether the defect in virulence was a

result of decreased replication on the epithelium of the foot and/or within the nervous tissue.










To determine whether the -83CRE virus exhibited a replication defect in nervous tissue,

mice were intracranially (i.c.) inoculated with the -83CRE virus to determine its virulence

following direct delivery to the CNS. In this assay direct inj section of 10 or 100 pfu of virus into

the brain of mice was performed. Mice were closely monitored for death and sacrificed once

CNS involvement was observed (determined by paralysis, erratic movement, and/or inability to

right itself). As shown in Table 3-2, the mutant -83CRE virus exhibited a slight decrease in

neurovirulence following i.c. inoculation when compared to 17syn+. These results suggest that

the -83CRE virus was attenuated in its ability to replicate in the nervous system, but it could kill

mice if delivered to the brain directly at higher doses.

The relative avirulence seen following footpad inoculation could have been due to a

cumulative effect of slightly less efficient replication at each node of the nervous system in

which the virus replicates on its path from the foot to the brain. It also was possible, however,

that the virus replicates less efficiently in the epithelium of the foot of the mouse and not just in

the nervous tissue. In order to differentiate between these possibilities a tracer study was

performed. Mice were infected via the footpad route and sacrificed at 8 hours, 2 days, or 4 days

p.i. Foot, spinal cord and DRG were assessed for relative numbers of viral genomes. As shown

in Figure 3-6A & B, replication in the foot is equivalent for all times examined. However, once

the -83CRE mutant virus reaches the DRG, replication is reduced by approximately 2.5- to 5.5-

fold relative to 17syn+, while replication in the spinal cord is reduced by approximately 5- to 8-

fold (Figure 3-6C, D). These results support the viral growth curve data, as well as the mouse

infection data, suggesting that the -83CRE mutant exhibits a reduced ability to replicate within

neurons.









The -83CRE Virus Contains a Second Site Mutation

Since the virulence phenotype observed for the -83CRE mutant was quite dramatic, it was

necessary to confirm that the -83CRE recombinant virus's phenotype was indeed due to the 8-bp

mutation in the LAT promoter. To do this the mutation was rescued by the transfection of wild-

type LAT promoter DNA with the -83CRE virus. If the -83CRE site was the only mutation in

the virus, restoration of the wild-type LAT promoter should restore virulence to the rescued

virus. Mice were inoculated via the footpad with varying doses (500, 5000, 50000 pfu/mouse) of

the wild-type, the -83CRE recombinant, or the rescue virus, F8-1. Little to no mortality was

observed for the F8-1 rescuant virus at any of the doses tested (Figure 3-7). The similar

virulence phenotypes of the F8-1 virus and the -83CRE virus suggested a second mutation

somewhere in the mutant virus's genome. Interestingly, when the F8-1 virus was assayed for

replication using a multi-step growth curve on RS cells, there appeared to be a delay in

replication efficiency, with decreased replication at 8 hours p.i. but levels similar to that of the

-83CRE and wild-type at the later times tested (Figure 3-8). This suggests that rescuing the

-83CRE mutation may have altered the ability of the virus to replicate on RS cells, but did not

rescue a second mutation that seems to primarily affect neuronal replication and virulence in

vivo. Ongoing experiments using subfragments of the viral genome to rescue the virulence

phenotype will map the site of the second mutation.

Mutation of the -83CRE Results in Wild-type HSV RNA Levels in RS Cells during Acute
Infection

To further analyze the -83CRE mutant, RS cells were infected at an m.o.i. of 0.01 with

either 17syn+ or -83CRE. Cells were harvested at acute times for RNA analysis. Random

decamer-primed reverse transcription reactions were followed by Taqman real-time PCR to

determine the relative quantities of select RNA transcript levels. Relative quantities of viral









targets were normalized to relative quantities of the cellular gene, GAPDH, to account for any

variations in cell growth or infectivity. As shown in Figure 3-9, there is no significant difference

in abundance of transcripts from any of the gene classes (immediate early, ICP4; early,

Polymerase; early-late, LAT; true late, glycoprotein C). Furthermore, inhibition of transcription

by treatment with cycloheximide or blockade of replication using phosphonoacetic acid showed

no difference in transcript accumulation between the wild-type and -83CRE viruses.

The -83CRE Virus Expresses LAT during Latency

Mice were infected with 400-500 pfu for latency. RNA was isolated from the dissected

DRG using guanidine thiocyanate isolation and was reverse transcribed using random decamers.

Relative quantities for the LAT 5' exon and ICP4 obtained through Taqman real-time PCR were

normalized to the relative quantity for the cellular RNA, XIST. As shown in Figure 3-10, the

mutant -83CRE virus expresses the LAT at a detectable level during latency. Additionally, the

relative values (HSV-1 polymerase normalized to cellular control XIST) determined for the

back-extracted viral DNA are 0.0183 + 0.0084 and 0.0228 + 0.0107 for 17s n+ and -83CRE,

respectively, indicating that levels of establishment of the -83CRE virus are similar to those seen

for wild-type 17syn+. These data indicate that mutation of the -83CRE site does not abolish

LAT expression during latency.

The -83CRE Virus LAT Promoter Region's H3 K9, K14 Acetylation Levels Are Similar to
Those of ICPO during Latency

In order to examine the chromatin profile of the -83CRE virus versus 17syn+, mice

infected with 400 pfu of virus were sacrificed and their DRG removed and processed for the

ChlP assay. Because CREs are able to recruit CREB binding protein (CBP), which is a known

histone acetyltransferase (HAT) (Ogryzko et al., 1996), anti-acetyl H3 K9, Kl4 was used to

determine whether mutation of the HSV-1 CRE affects the acetylation levels of the LAT










promoter and/or the LAT enhancer. It was previously shown that the region encompassing the

LAT promoter and 5' exon is enriched in H13 K9, Kl4 acetylation relative to lytic genes (Kubat

et al., 2004). As shown in Figure 3-11, that is not observed for the -83CRE virus. In fact, the

LAT promoter region is approximately as hypoacetylated as the lytic gene, ICP0. This suggests

an abrogation of the ability of CBP, or some other factor affecting transcriptional

permissiveness, to bind the LAT promoter when the -83CRE site is mutated.

The -83CRE Virus Reactivates from Latency in the Rabbit with Similar Efficiency as Wild-
type 17syn+

Rabbits were infected via the ocular route at a dose of 200,000 pfu/eye of wild-type

17syn+ or -83 CRE. Slit lamp examination of the rabbit eyes at three, five, and seven days post-

infection revealed no significant difference in pathology between the viruses (Table 3-3).

Furthermore, both viruses had similar mortality, with 12/26 rabbits (46%) surviving the 17syn+

infection and 10/20 rabbits (50%) surviving infection with the -83CRE virus. To assess

reactivation efficiency, once the viral infections became latent, the rabbit eyes were subj ected to

epinephrine iontophoresis and swabbed for eight days post-induction. There was no significant

difference in reactivation, with the -83CRE recombinant reactivating at near wild-type levels

(P=0.4, Table 3-4).

Discussion

Mutation of the -83CRE Is Unfavorable for Recombination

The construction strategy of the -83CRE recombinant should have essentially rescued the

LAT promoter deletion of the parental 17APst-Stuffer by replacing the bacterial stuffer fragment

with a viral DNA fragment (Pst-Pst fragment) containing the LAT transcriptional start site, as

well as a mutated -83CRE site. Detection of the bacterial stuffer fragment in the parental virus

would suggest that the recombination did not occur. After eight rounds of purification, the









recombinant was fully positive for the Pst-Pst fragment but also fully positive for the bacterial

stuffer fragment. This suggested that both regions were present in the recombinant virus, and

this hypothesis was confirmed when the region was amplified by PCR (Figure 3-2).

Recombination, therefore, appeared to occur in only one of the two copies of LAT existing in the

viral genome, suggesting that the wild-type -83CRE site is important in virus viability. In other

words, a -83CRE recombinant with the mutation in both copies of LAT might prevent normal

replication from occurring. The LAT is anti-sense to ICPO and the 3' ends of the two transcripts

overlap. Because of this, one might hypothesize that the -83CRE site normally acts as a

transcriptional repressor to control LAT expression at inappropriate times, i.e., times when ICPO

is expressed. When the site is mutated, the virus can no longer replicate because of LAT's

interference with ICP0. To address this possibility, the virus was plaque purified on L7 cells. L7

cells are a Vero-based cell line that was stably transfected to express ICPO (Samaneigo et al.,

1997). Interestingly, after only three to four rounds of plaque purification, all plaques were

positive for the Pst-Pst fragment while none were positive for the bacterial stuffer.

Plaque purification of the -83CRE virus may have been successful on L7 cells and not on

RS cells for the reason mentioned above. Since the need for ICPO complementation was only

seen during the plaque purification stage, and not in later experiments, LAT regulation is

probably important during recombination. It may be possible that the -83CRE site acts as a

repressor of LAT, acting to prevent LAT transcription from occurring at the same time as ICP0.

If so, when the -83CRE site is mutated, expression of ICPO in transrt~t~rt~t~rt~t~rt~ by the L7 cells is necessary

to overcome the effects of LAT misregulation.

Alternatively, a need for ICPO in recombination may be caused by the still unidentified

second site mutation. If the second mutation affects a region of the virus that is involved in









recombination and ICPO must be provided in trains to overcome the effect, it may suggest that

ICPO and the second site interact during recombination or more likely, that the second site

mutation is in ICPO and that ICPO itself is important in recombination.

The -83CRE Recombinant Contains a Second Site Mutation that Contributes to the
Avirulence Phenotype

When the -83CRE mutant virus was rescued using the wild-type LAT promoter, the

avirulence phenotype was not rescued. The mortality rate of the rescuant-infected mice was very

low, indicating the presence of another mutation somewhere in the -83 CRE mutant' s genome.

This other mutation, therefore, is likely a maj or contributor to virulence of HSV-1.

The second site mutation in the -83CRE recombinant may have arisen during the viral

construction phase. If the mutation in the -83CRE site was unfavorable to viable virus

production, a second compensatory mutation may have occurred, which ultimately allowed the

recombinant to be made. An example of a similar situation exists with neurovirulence gene

y34.5 viral recombinants. In eukaryotic cells part of the host defense against viral infection is the

activation of protein kinase R (PKR) in response to double-stranded RNA. PKR induction in a

cell causes eukaryotic translation initiation factor 2 (elF-2a) to be phosphorylated, which

ultimately shuts down protein synthesis. In wild-type HSV-1, the y34.5 gene product can

dephosphorylate elF-2a to prevent cellular shutdown of protein synthesis (He et al., 1997). As

expected y34.5 mutants are unable to prevent cellular inhibition of protein synthesis (Chou and

Roizman, 1992). Rescue of the y34.5 mutation led to the isolation of mutants with a second site

mutation in the U 1 1 gene that causes restoration of the PKR suppressor phenotype observed in

wild-type HSV-1 (Mohr and Gluzman, 1996). U 1 1 interacts with PKR if present before PKR

induction to block elF-2a phosphorylation and therefore, allows protein synthesis to proceed and









the virus to function (Cassady et al., 1998). The second site mutation in the -83CRE mutant

could be functioning in a similar manner, compensating for the -83CRE mutation.

While the second site mutation in the -83CRE virus remains unmapped, one can speculate

as to its location. Potentially, the mutation could lie somewhere in ICPO, affecting its normal

function. In ICPO-negative mutants, growth in cell culture is inefficient at low m.o.i., but

replication is restored to near wild-type levels at high m.o.i. (Sacks and Schaffer, 1987; Chen and

Silverstein, 1992). While the -83CRE recombinant did not show the same effect as ICPO

mutants on RS cells, the results from the growth curve assays on Neuro-2A cells suggest

multiplicity-dependent growth in neuronal cells. Recall that the -83CRE virus replicated less

efficiently than wild-type on Neuro-2A cells at a low m.0.i., but the mutant replicated as well or

slightly better than wild-type at the high m.o.i. when grown on Neuro-2A cells. This may

indicate that the -83CRE recombinant is functionally deficient in ICPO in neuronal cells, possibly

due to a lack of necessary interplay between effects of the -83CRE mutation, ICPO, and some

neuronal-specific factor. When ICPO is present at high levels, growth in Neuro-2A cells can

occur normally. The -83CRE recombinant appeared to grow more efficiently than wild-type

during early infection of RS cells at a low m.o.i., suggesting that the -83CRE mutation may

compensate for a mutated form of ICP0. The animal experiments performed, in which various

tissues were assessed for relative viral DNA levels, support this hypothesis (Figure 3-6). When

the -83CRE initially infected the mouse footpad, DNA levels were similar between wild-type

and the mutant, but levels of the mutant viral DNA were decreased relative to wild-type in the

DRG and spinal cord. This could be due to a lower effective dose of the virus in those tissues;

perhaps the -83CRE mutation is no longer an effective means of compensation for whatever

defect is present in ICP0. In other words, when the mutant virus is applied to the footpad at a










high dose, it would not appear to act like an ICPO mutant, in which replication is inhibited at a

lower m.o.i. Once the -83CRE virus enters the neuronal tissues, however, the amount of viral

production begins to drop. This may be caused by an inability of the mutant virus to utilize some

neuronal factor properly and has the end result of decreasing the effective viral dose to the

neuronal tissues to one that is not compatible with efficient growth by an ICPO-defective virus.

In the mouse survival experiments, the -83CRE virus was severely attenuated for

virulence, corresponding to the finding that ICPO deletion mutants display decreased

pathogenicity in some animal models (Gordon et al., 1990). What is puzzling, however, is the

fact that the -83CRE virus established latency and reactivated from latently-infected rabbits with

similar efficiency as wild-type 17syn+, unlike characterized ICPO mutants (Wilcox et al., 1997;

Halford and Schaffer, 2001). When the chromatin profile was determined for the latent -83CRE

in mouse DRG, the LAT promoter and the ICPO promoter had similar levels of H13 K9, Kl4

acetylation, which is a marker of transcriptional permissiveness, and both of these regions were

less acetylated than the LAT 5'exon. Perhaps the chromatin profile of the -83CRE mutant

permitted establishment of latency and reactivation. In other words, even though ICPO was still

hypoacetylated relative to the 5' exon, it may have still been slightly permissive for transcription

(which would not be apparent from the qualitative results of the ChlP assay), and this would

have allowed reactivation to occur. Marker rescue experiments will aid in determining the site of

the second mutation.

Cell-Specific Factors May Interact with the -83CRE Site to Convey Neuronal Tropism to
HSV-1

The results from the viral growth curves and the mouse tracer study indicate differential

growth efficiencies of the -83CRE recombinant on epithelial cells than on neuronal cells.

Specifically, the mutant replicates more efficiently than the wild-type virus on epithelial cells,









while it does not grow as well as wild-type on neuronal cells. This may also explain why the

-83CRE is attenuated when mice are infected by the footpad infection route. It seems likely that,

although the mutant replicates as efficiently as wild-type at the footpad, the efficiency of

replication slows once the mutant reaches the spinal cord and becomes even less efficient once

reaching the DRG. By the time it reaches the brain, which is the site of encephalitic infection

that leads to mortality in mice, there is little infectious virus left to cause damage.

When the -83CRE virus was used to infect rabbits, the virus did not show the virulence

deficit that was observed for mice. The rabbits displayed similar levels of corneal pathology as

wild-type, suggesting robust replication in the corneal epithelia by the -83CRE. Because the site

of infection (cornea) in the rabbit is close to the site of latency (TG), the virus has less distance to

travel and therefore, the -83 CRE virus might also replicate less effectively in neurons in the

rabbit, as was seen for the mouse, but because of the proximity to the brain, may still produce

enough infectious virus to cause death. Experiments to determine viral yields in epithelia versus

neuronal tissue during acute infection in the rabbit are in progress.

Mutation of the -83CRE Site Does Not Affect Latency and Reactivation

While mutation of the -83CRE site affected the acute infection of HSV-1, it had no

significant effect on the establishment of latency, nor did it affect the ability of the virus to

transcribe LAT. This suggests that the -83CRE site, in conjunction with the second site

mutation, does not play a role in recruitment of transcription factors to the region during latency.

However, when the chromatin profile was determined, the -83CRE mutant virus displayed a

profile unlike that seen for wild-type. During latency the -83CRE virus's LAT promoter was

less enriched than the 5' exon/enhancer region and was also less enriched than in wild-type

17syn+. This hypoacetylation was almost as low as that of the ICPO promoter, suggesting a









decrease in transcriptional permissiveness in the -83CRE LAT promoter resulting from a loss of

the binding site. However, the LAT was still detected by RT-PCR of latently-infected DRG.

Because CRE sites are stress-responsive elements, it was originally hypothesized that the

LAT promoter's CREs would play a role in reactivation. However, the -43CRE displayed only

an intermediate defect in reactivation when tested in rabbits (Bloom et al., 1997), while the

-83CRE virus reactivated at the same frequency as wild-type HSV-1. It remains possible that the

two elements act in concert with each other to control reactivation, since the deletion of both

elements in 17APst results in a virus that does not reactivate from latency in the rabbit (Hill et al.,

1990). However, since the deletion in 17APst is 202 bp, many important factors besides the

CREs are deleted, which is likely the cause of the reactivation-negative phenotype. In order to

address possible interactions between the two LAT promoter CREs without considering the

influence of the other binding sites in the promoter, a double CRE mutant would need to be

created. While not detailed in this dissertation, both the -43CRE and -83CRE were mutated and

transfected with HSV-1 DNA in order to create a recombinant with both CREs mutated

(CREDBL). Like the -83CRE, the CREDBL remained single-sided throughout the plaque

purifications on RS cells. Unlike the -83CRE virus, the CREDBL was unable to be purified on

L7 cells. Only about 50% of recombinants were positive, although single-sided, and none

became pure after several more rounds of purification. This, coupled with the -83CRE viral

construction difficulties, implies a need for the wild-type -83CRE in at least the acute, replicative

stage of the viral lifecycle, during which recombination would occur. Since the -43CRE

recombinant was constructed by Bloom et al. (1997) with little difficulty, this element is likely

not as critical as the -83CRE for lytic viral growth.









The Wild-Type -83CRE May Control LAT during the Acute Infection

While the -43CRE showed a slight effect on reactivation, there was no significant effect on

reactivation that resulted from mutation of the -83CRE. Instead, the major observations--

decreased neuronal replication and attenuated virulence in the mouse--are linked to the acute

HSV-1 infection. Since it was previously reported that the -83CRE binds the repressive CREB-2

(Millhouse et al., 1998), the site's main function may be to control LAT. When HSV-1 enters a

cell, the cAMP pathway may be induced, since the entry of a virus into a cell is undoubtedly a

stressful event. Various CREB family members could be produced, including CREB-2. When

this is present at the LAT promoter' s -83CRE, the promoter would be repressed. However, in

the event that the site is mutated and CREB-2 binding is abrogated, perhaps LAT is no longer

controlled and would interfere with ICPO to affect the acute stage of infection. For this reason,

the -83 CRE recombinant was not able to be constructed with the mutation in both repeats of the

LAT. Instead, a second site compensatory mutation was necessary that allowed for the viability

of the -83CRE virus. This mutation may have occurred in ICPO, possibly affecting some

function that normally controls ICPO expression. If LAT interference with ICPO by the -83CRE

mutation needed to be overcome, the likely solution for the virus might be to upregulate ICPO,

providing the -83CRE recombinant with a similar environment as that encountered when grown

on L7 cells. By characterizing the second site mutation and determining whether it really does

occur in ICPO, the importance of the LAT promoter -83CRE site and how it might interplay with

the region of the second mutation can be uncovered.












Table 3-1. Survival of mice (number remaining/number infected) infected via the footpad with
either 17syn+ or -83CRE.
Dose (pfu) 17syn+ survival -83CRE survival
500 3/10 9/10
5,000 1/10 9/10
50,000 0/10 9/10

Table 3-2. Survival of mice (number remaining/number infected) inoculated intracranially with
either 17syn+ or -83CRE.
Dose (pfu) 17syn+ survival -83CRE survival
10 1/5 4/5
100 0/5 0/5

Table 3-3. Slit lamp examination (SLE) scores of rabbit corneas at 3, 5, or 7 days post-infection.
Day post-infection 17syn+ average SLE score -83CRE average SLE score P-value*
3 3 +1.1 3 +1.2 0.82
5 2.8 + 1.1 2.7 + 1.0 0.5
7 2.3 + 1.1 2.3 + 1.1 0.98
* P-value calculated using the Mann-Whitney Rank Sum test.

Table 3-4. Reactivation of rabbits post-epinephrine induction.
.im Percent reactivated eyes Percent reactivated swabs
(Total positive/total eyes) (Total positive/total swabs)
17syn+ 54.5% (6/11) 26.1% (23/88)
-83CRE 78.6% (11/14) 32.1% (36/112)













LLI


C 3C,
Cr)
tc~X
~xlaj
r- Do r- 2;





~W 3n,


Figure 3-1. Diagram of PCR primer locations used in verification of -83CRE mutant virus.
Recombination fragment for virus construction is shown as a gray bar, with the
-83CRE site shown as a black box. The LAT transcriptional start site is indicated by
an arrow. Primers are designated by arrows and U, Upfragment (nt 118,305-
118,325), or D, Downfragment (nt 119,179-119,202).


W
d
U
z


A


Figure 3-2. Analysis ofPCR products amplified from dot blot purification. A) The recombinant
virus in an early round of purification shows products corresponding to both the
parental and wild-type (17syn+) virus. Diagram of the -83CRE recombinant genome
shows the two copies of the LAT (arrows) and indicates the presence of the stuffer
(green) and the mutation (red "X"). B) The recombinant virus PCR product size
corresponds to that of the wild-type virus (17syn+) in purification on L7 cells. Note
the presence of a single band. Parental virus, 17APst, is also shown. NTC, no
template control.


41~

IlrrlC














le49 .


lo45


Hours post-infecha


p109 -


IpC-


Hurps post-infechan


lo49S


1.48


Hor post-ifeda


Figure 3-3. Multi-step viral growth curves. 17syn+ (wild-type) or -83CRE (recombinant) were
inoculated at an m.o.i. of 0.01 to allow for multiple rounds of replication on A) RS

cells, B) Neuro-2A cells, and C) L7 cells.


-e- 17ryxi


-*- 17syri-
















le+8 --







-*- 17lsyn+
le+6 ]-I--- -8TjRE



le+5
0 4 12 24
Homns postmbeetioa



B 1+




le+7





le+6
-*-17syn+
-w-+- -gTRE



le+5
0 4 12 24

Hom potmfcto


Figure 3-4. Single-step viral growth curves. 17sym+ (wild-type) or -83CRE (recombinant) were
inoculated at an m.o.i. of 5 to allow for synchronous infection of all cells and a single
round of replication on A) RS cells and B) Neuro-2A cells.








































IIIIIII I


3 1 3 ] i 5 1 7 9 9 10 11 12 13 14 15 11 17 18 19 20 31


100


10 -


0 1 3 ] 4 5 1 7 $ 9 10 11 12 13 14 15 11 17 18 19 20 31


100 -


>~---o------o--o---

o ---o---o-- o--o -- ---o---o---o---0


-e- 17rsyzi
20 -I ---0--- CICHE




0 IIIII


7 8 9 10 11 12 11 14 15 11 17 18 19 30 21


Figure 3-5. Percent survival over the course of acute infection of mice (10 per group) infected

with either 17syn+ or -83CRE at A) 500 pfu, B) 5,000 pfu, or C) 50,000 pfu.


-e- 17rysi-
---0-- -MCII


-*- 17rysi-
...0- -mCEn

















04





01



250
















10U4

IOU2


150

loo

Iso

too

ro

0

160

80

60

40

20

0

16

12

8

4

0


I


a
9
a



C

B
Q
a
9





I> ,
P
e
L
1


d


Figure 3-6. Relative viral genomes (Pol, HSV-1 polymerase normalized to cellular gene, Xisi)
for 8 hours, 2 days, or 4 days p.i. of mice. Mice were infected via the footpad with

either 17syn+ or -83CRE. A) 8 hours p.i. foot samples (n = 4 mice/virus/time), B)

foot samples for 2 days p.i. (left panel) and 4 days p.i. (right panel), C) DRG 2 days

p.i. (left panel) and 4 days p.i. (right panel) (n = 5 mice/virus/time), D) Spinal cord 2

days p.i. (left panel) and 4 days p.i. (right panel) (n = 5 mice/virus/time).


I;
*n+
gl
r
t
a








E
E










g
D































-*-17sytt+
....g... -83CRE
--T- F8-1


0 ;1 234 5 6


8 9 10 11 12 13 14 15 16 17 18 19 20 21

Days post-in etion


fT-~-~f ff'P17f T



C-3-04-000-00


-*-- 17sytt+
...g... -83CRE
--T- F8-1


0 1 3 4 56 7


8 9 10 11 12 13 14 15 16 17 18 19 20 21

Days post-infetion


--+-- 17syn+
....o.... -83CRE
-Y 7-1


If

Ib~tttt-


0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Da~ys posit-infetion


Figure 3-7. Percent survival of mice infected with A) 500 pfu, B)

17syn+, -83CRE, or F8-1 rescuant virus.


5,000 pfu, or C) 50,000 pfu of


'ITf





































0 24


Time! (hours p.i.)


Figure 3-8. Multi-step growth curve (m.o.i. 0.001) on RS cells for 17syn+, -83CRE, and F8-1.


-*-- 17 syn+
-g- -83CRE
le8 --y- F8-1














Untreated


CHX


PAA


Time (oms post-inftaion)


Time (ours post infection)


O 5




8001



Time (ourspost-infeion)


Time (ours post-mffecton)


Tune (hurs ost infection)


Figure 3-9. Relative RNA transcript levels (target normalized to cellular control, GAPDH)
during acute infection ofRS cells. A) Immediate-early gene, ICP4, B) early gene,
HSV-1 polymerase, C) late gene, LAT, and D) late gene, glycoprotein C (gC). Left

panel, no treatment. Middle panel, addition of 50 Cpg/ml CHX to cells along with
infecting inoculum. Right panel, addition of 400 Cpg/ml PAA to cells with infecting
inoculum. Black bars, 17syn+. Gray bars, -83CRE recombinant.


00W20 L0020



MI hII M
1 8
Tun (hous potifcin ie(ar otifcin


B








Time (oms post-infection)


ooose acces



8~ r4
Time (oms post-infection) Tune (ours post ffetlon)


D-




g~ t


Time (homspost-mnfction)


Tune (hours st-infection)






























5
=


oS


1~
s
B
P;I


Figure 3-11i. The LAT promoter of the -83CRE mutant virus is decreased in histone H3 K9, Kl4
acetylation relative to wild-type. A) 17syn+, B) -83CRE recombinant. Relative
bound, B, to unbound, U, ratios normalized to those for Xist cellular control are
shown for the LAT promoter (L. Pro.), the 5' exon, the ICPO promoter, and ICP27.


a
t~2 I


Figure 3-10. LAT expression during latency in mouse DRG infected with the -83CRE mutant
virus. RNA was isolated from DRG of latently-infected mice and reverse transcribed
using random decamers. PCR was performed using LAT 5' exon primers.


g5' eman
AICH)
x ICP27









CHAPTER 4
OVERALL DISCUSSION

While the HSV-1 LAT region is known to facilitate reactivation, the exact mechanism

involved is not known. The overall focus of this dissertation, therefore, was to investigate

regulation of the LAT, particularly at the level of the LAT promoter. Findings suggest that (1)

an element or elements in the core LAT promoter are essential for a latent chromatin profie that

is compatible with reactivation, (2) epinephrine-induced reactivation of latently-infected rabbits

does not cause the same effects on chromatin as explant-induced reactivation of mouse DRG, (3)

the -83CRE site of the LAT promoter is dispensable for establishment of latency, ability to

express LAT, and capacity to reactivate, even though the latent chromatin profie differs from

that of the wild-type virus, and (4) a compensatory mutation generated during the construction of

the -83CRE recombinant appears to dramatically affect virulence in the mouse and may be

important in regulation of the lytic infection. These data support the developing view that the

LAT promoter may be a part of a complex regulatory switch that modulates gene expression in a

tissue-specific manner both during the acute and latent periods of HSV-1 infection.

The LAT and Chromatin

During latency transcription from the HSV-1 genome is not repressed through DNA

methylation, but instead, histone tail modifications may be a component of gene regulation

(Kubat et al., 2004a). In the mouse the LAT region of the latent viral genome shows enrichment

in acetylated histone H3 K9, Kl4, a marker of transcriptional permissiveness, relative to lytic

genes (Kubat et al., 2004a; Kubat et al., 2004b). When the LAT promoter is deleted and the

virus is unable to transcribe the LAT, the same effect is observed, suggesting that the LAT

region contains elements that direct the transcriptionally permissive histone modifications

independently of Pol II activity through this region. During early times post-explant of murine









DRG infected with wild-type virus, there is chromatin remodeling of the LAT region to a more

transcriptionally non-permissive state and decreased LAT levels between 2 and 3 hours post-

explant (Amelio et al., 2006). A maj or goal of this dissertation project was to extend the results

obtained for the mouse to the rabbit eye model, which more closely mimics clinical reactivation

of HSV-1 in humans.

In the rabbit, the ability to express at least some regions of the LAT correlates with the

ability to reactivate from latency. When the chromatin profie was assessed for a LAT-positive

(wild-type) and a LAT-negative (promoter-deletion mutant) virus, two different profies were

observed, unlike what was seen in the mouse. While the wild-type virus displayed a similar

latent profie to that of the latently-infected mouse, the LAT-negative virus displayed

dramatically more enrichment in dimethylated H13 K4, a marker of transcriptional

permissiveness, in the region just upstream of the deletion than the 5'exon/enhancer. Levels of

dimethylation in the LAT promoter region were also much higher than that of wild-type, even

though the 5'exon/enhancer region showed similar levels. This suggests the presence of a

repressive element in the native core LAT promoter that might prevent increased transcriptional

permissiveness of the LAT region at inappropriate times, such as during lytic infection.

Additionally, because LAT promoter deletion mutants are severely deficient in reactivation in

the rabbit, the difference in chromatin profiles between the mutant and the wild-type virus may

implicate a requirement for establishment of a certain chromatin profile for normal reactivation

and also suggests that deletion of the LAT promoter causes a loss of a regulatory element

required for wild-type reactivation.

When the chromatin profiles of wild-type HSV-1 and LAT promoter mutant, 17APst, were

assessed following adrenergic induction of reactivation in the rabbit ocular model, no change in









chromatin was evident for either virus between 0, 1, 2, and 4 hours post-induction, times

corresponding to those tested in the mouse explant experiments. This finding suggests that

chromatin remodeling is not critical to LAT-dependent reactivation or that chromatin remodeling

in response to mouse DRG explant is a much faster process than what occurs in the rabbit. For

example, since the epinephrine is administered to the eye and reactivation occurs in the ganglia,

it might take some time for the epinephrine to reach and stimulate all cells in the ganglia, while

in explant-induced reactivation, a uniform stressor (ganglion removal from the animal) might

stimulate cells to reactivate very rapidly.

The deletion of the core LAT promoter in the 17APst mutant is 202-bp in size. This

suggests that a number of binding sites and cis-elements were removed, some of which could

normally contribute to controlling the level of transcriptional permissiveness.

LAT Regulation through Promoter Function

To investigate a possible regulator of the LAT promoter, a cAMP response element,

located 83 nucleotides upstream of the LAT transcriptional start site (-83CRE) was examined. A

recombinant virus with mutation of the -83 CRE was created and analyzed for alterations to the

acute, latent, and reactivation phases of the viral lifecycle. The -83CRE recombinant was

ultimately found to contain a second mutation, which may have been a compensatory mutation

that allowed an otherwise unviable recombinant to replicate. During acute infection, the mutant

virus displayed attenuated virulence in mice that were infected via the footpad, and in cell culture

the virus showed increased replication in fibroblasts and decreased replication in neuronal cells,

suggesting a cell-type specific effect of the -83CRE site (or the compensatory mutation) in viral

replication. The -83CRE recombinant virus was able to establish latency, express LAT, and

reactivate. Interestingly, during latency in mice, the -83CRE mutant virus displayed decreased

transcriptional permissiveness at the LAT promoter relative to the wild-type virus. This is in









contrast to the findings for 17APst in the rabbit, in which deletion of the entire promoter

(including the -83CRE site) caused dramatically increased transcriptional permissiveness. Thus,

the wild-type -83CRE may be important in regulation of the LAT promoter, probably by

interacting with another nearby element as well as cellular factors, to maintain the configuration

of the LAT region in a reactivation-compatible state. Removal of the -83CRE causes a

decreased transcriptional permissiveness of the LAT promoter yet the recombinant virus still

reactivates from latency in the rabbit. Since there is another mutation in the recombinant, that

second site may contribute to the chromatin configuration. For example, iflICPO is mutated to a

more active form, control of latency might not be as tightly controlled as in the wild-type virus;

thus, the -83CRE mutant virus can still reactivate because it is less repressed. Future

experiments should address these questions.

Findings suggest that the -83 CRE is important in acute phase regulation of HSV-1, likely

in concert with some other elements) of the genome. If the wild-type -83CRE binds a

repressive protein to control LAT expression during the lytic infection, deletion of the element

would cause misregulation of the LAT, potentially interfering with ICPO transcription. If this

occurred during the construction phase of a mutant -83CRE virus, a second mutation in ICPO

may have occurred to overcome the problem. Characterization of the second mutation in the

-83CRE recombinant is ongoing and should provide information about interplay between the

LAT promoter and other elements important to the viral life cycle.

A Model for Reactivation

A model has been proposed for steroid receptor function in which random and transient

interactions between various factors and a promoter might not actually result in transcription;

instead, since those events may be part of a sequential process, in which promoter modification

and secondary recruitment of other factors ultimately lead to transcription, ChlP experiments









would indicate only some of the events in a population at a particular time without giving a sense

of the dynamic processes occurring in single cells (reviewed in Hager et al., 2006). In other

words, the binding of receptor complexes, and probably other transcription factors, is a cyclical

process; protein-DNA binding occurs without being stable and long-term, but the end result is

usually promoter activation. Support for this comes from experiments performed on single

living cells using UV laser crosslinking technology to monitor chromatin remodeling at a

promoter; these studies indicated cycle times were less than a minute (reviewed in Hager et al.,

2006). The approaches used in the experiments performed in this dissertation used ChlP, thereby

limiting observations to events occurring in the collective group of cells in a ganglion.

Perhaps the regulation of HSV reactivation could be modeled like the dynamic process that

regulates steroid receptor activation, and at any given time, single "latently-infected"' cells are

actually producing lytic viral transcripts. Support for this comes from a study performed by

Feldman et al. (2002), in which ganglia from latently-infected mice were assayed by in situ

hybridization for the presence of lytic gene expression and viral replication. Assessment of

thousands of neurons led to the finding that a small number of individual neurons actually

express lytic viral genes and replicate viral DNA, even though the ganglia as a whole is

considered latently-infected. The authors also found indication that the host inflammatory

response may prevent the stray neurons from yielding infectious virus (Feldman et al., 2002).

Perhaps true reactivation does not actually occur until a group of those cells is synchronously

transcribing lytic genes, and enough virus is produced to be infectious. Activation of the cAMP

pathway, or another stress-inducible pathway, by a stress stimulus may be sufficient to not only

trigger the synchronous initiation of reactivation in a larger number of cells, but also to create the









correct environment (i.e., temporary depression of the local host response) for efficient

production of abundant virus.

The entire process of establishment of and reactivation from latency is clearly complex.

Findings can vary from system to system and viral strain to viral strain, possibly due to different

types of reactivation. If regulation at the level of chromatin is important, maybe a specific

chromatin configuration must be established that is compatible with reactivation. If cis elements

in the LAT rcr region are important, maybe timing and synchronicity are critical for reactivation.

However, it may be possible that both of these, as well as other regulatory mechanisms, play a

role in preventing aberrant transcription of lytic genes and controlling reactivation. HSV-1's

ability to persist indefinitely in a host, undetected by the immune system, and periodically

reactivate to spread to a new host suggests a very tightly regulated system that may include

several ways for reactivation to occur. Work in the field of latency and reactivation will

undoubtedly provide interesting and numerous pieces of the puzzle for years to come.





APPENDIX
REAL-TIMVE PCR PRIMVER/PROBE SEQUENCES

Table A-1. Real-time PCR primer/probes.


Target
LAT Promoter
(NC_001806: 118,263-118,323)

LAT 5' Exon
(NC_001806: 119,326-119,397)

ICPO Promoter
(NC_001806: 124,494-124,578)

ICP27/UL54
(NC_001806: 113,945-114,034)

ICP4
(NC_001806: 147,941-148,025 )


Sequence (5' to 3')
Forward--CAA TAA CAA CCC CAA CGG AAA GC
Reverse-TCC ACT TCC CGT CCT TCC AT
Probe-TCC CCT CGG TTG TTC C
Forward--GGC TCC ATC GCC TTT CCT
Reverse-AAG GGA GGG AGG AGG GTA CTG
Probe-TCT CGC TTC TCC CC
Forward--CCG CCG ACG CAA CAG
Reverse-GTT CCG GGT ATG GTA ATG AGT TTC T
Probe-CTT CCC GCC TTC CC
Forward--GCC CGT CTC GTC CAG AAG
Reverse-GCG CTG GTT GAG GAT CGT T
Probe-CGAG CAC CCA GAC GCC
Forward--GAC GGG CCG CTT CAC
Reverse-GCG ATA GCG CGC GTA GA
Probe-CCG ACG CGA CCT CC
Forward--AGA GGG ACA TCC AGG ACT TTG T
Reverse-CGAG GCG CTT GTT GGT GTA C
Probe-ACC GCC GAA CTG AGC A
Forward--GCA CCA CCA ACT GCT TAG C
Reverse-CCT CCA CAA TGC CGA AGT G
Probe-CTG GCC AAG GTC ATC C
Forward--GCT CCA GAA ACC TGA GAA AAC ATG
AT
Reverse-TGG AGA AAA GCG CAA TCT TCC T
Probe-TTC GGC AAA TGC ATC CAA
Forward--GCT CTT AAA CTG AGT GGG TGT TCA
Reverse-GTA TCA CGC AGA AGC CAT AAT GG
Probe-ACG CGG GCT CTC CA
Forward--CTC AAG AAA TCT AAC CCC TGA CTC A
Reverse-GCG GGA CAG GCT GAG A
Probe-CCA GGG CCT CAC CAC C


HSV polymerase
(NC_001806: 65,801


-65,953)


Rabbit GAPDH


Rabbit centromere



Mouse Xist
(NR 001463: 857-925)

Mouse APRT










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BIOGRAPHICAL SKETCH

Nicole Giordani maj ored in microbiology at the University of Arizona in Tucson, Arizona

from August 1998 to December 2001. During this time, Nicole was part of the Undergraduate

Biology Research Program (UBRP) and performed research on the alpha subunit of the RNA

polymerase of Euglenoids under the supervision of Richard Hallick, Ph.D. After receiving her

Bachelor of Science degree in December 2001, Nicole applied to graduate school and began the

University of Florida' s Interdisciplinary Program (IDP) in August 2002. Upon earning her

Ph.D., Nicole performed post-doctoral work in an academic setting.





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1 REGULATION OF THE HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) LATENCYASSOCIATED TRANSCRIPT (LAT) By NICOLE V. GIORDANI 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 2007

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2 2007 Nicole V. Giordani

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3 ACKNOWLEDGMENTS First and foremost, I thank my parents and fa mily for encouraging me to follow whatever career path I chose. I wouldn’t be where I am today without their support and love, and I will always be grateful. I thank my undergraduate mentor, Richard Hal lick, for giving me th e chance to get my start in a lab. In addition, I thank his post-doc toral associate, Elena Sheveleva, for her patience and kindness during her days of working with me. I especially thank Dave for his guidance over th e past few years. He has taught me a lot about many aspects of science and has always pr ovided encouragement, even when I was not sure that I could do it. I have always appreciated the enthusiasm with which he approaches science, and I hope some of it has worn off on me. I thank all of the graduate students (Niki, Je rry, Anne, Lee, Tony, Zane, and Dacia) that I worked with over the years for each teaching me something and helping me whenever I needed it. To list what I have learned from each of them would be impossible, since the knowledge they have passed on to me has been insurmountable. I also thank Dacia for cri tical reading of this dissertation and for helping me to deal with the st ress. I thank Peterjon (“the best tech ever”) for all of his computer and cell culture assistance and for helping me with so many other things here and there. I thank Joyce for he r expertise with cloning and for al ways offering to help when she sees the work piling up. I thank my committee—Jorg Bungert, Rich C ondit, Bert Flanegan, and Dick Moyer—for their helpful suggestions and for keeping me on track. Many other people have helped shape me as a scientist, and I thank all of them.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION................................................................................................................. .12 Basic Overview................................................................................................................. ......12 Productive Infection........................................................................................................... .....12 Latency and Reactivation....................................................................................................... 14 Animal Models................................................................................................................14 The Latency-Associated Transcript (LAT).....................................................................16 The LAT during lytic infection................................................................................17 The LAT during latency and reactivation................................................................17 LAT Promoter and LAT Transcription...........................................................................19 Eukaryotic Epigenetic and Transcriptional Regulation..........................................................23 Epigenetic Regulation.....................................................................................................23 Promoter Elements and Transcriptional Regulation........................................................26 Summary........................................................................................................................ .........27 2 CHROMATIN CONFORMATION OF THE LATENT HSV-1 GENOME IN RABBITS........................................................................................................................ .......31 Objective...................................................................................................................... ...........31 Background and Previous Findings........................................................................................31 Materials and Methods.......................................................................................................... .34 Rabbit Infections.............................................................................................................3 4 ChIP Assay..................................................................................................................... .34 Taqman Real-Time PCR Analysis..................................................................................35 Results........................................................................................................................ .............36 The Chromatin Profile of the Latent HSV Genome in Rabbits Latently-Infected with Wild-Type HSV-1 Is Similar to that Observed for Latently-Infected Mice........36 Deleting the Core LAT Promoter Results in Increased H3 K4 Dimethylation of the LAT Promoter Region in Latently-Infected Rabbits...................................................36 Neither 17 syn + nor 17 Pst Display Dynamic Changes in the LAT Region in Response to Epinephrine Induction of Latently-Infected Rabbits...............................37 Relative H3 K4 Dimethylation Levels of the LAT Promoter Region in 17 Pst Are Higher than Those of Wild-type for All Times Examined..........................................37

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5 Discussion..................................................................................................................... ..........37 Kinetics of Chromatin Remodeling.................................................................................37 A Repressive Element in the LAT Promoter...................................................................38 3 INVESTIGATION OF THE ROLE OF A LAT PROMOTER cAMP RESPONSE ELEMENT (CRE) IN REACTIVATION..............................................................................44 Objective...................................................................................................................... ...........44 Background and Previous Findings........................................................................................44 Materials and Methods.......................................................................................................... .49 Plasmid Generation, Mutagenesis, and Purification........................................................49 Cells and Viruses.............................................................................................................5 0 DNA Isolation for Transfections.....................................................................................51 Virus Construction and Plaque Purification....................................................................51 PCR Analysis................................................................................................................... 54 Growth Curves.................................................................................................................5 4 Mouse Survival Assay.....................................................................................................55 Intracranial Inoculation as an Assay for Neurovirulence................................................55 DNA Extraction and Analysis of Course of Infection.....................................................55 RNA Isolation and Reverse Transcription for Acute RNA Levels in Cell Culture........56 Taqman Real-time PCR Analysis....................................................................................57 RNA Isolation and Reverse Transc ription for Explant Studies.......................................57 ChIP Analysis.................................................................................................................. 58 Rabbit Reactivation.........................................................................................................59 Results........................................................................................................................ .............60 The -83CRE Recombinant’s Replication Is Altered during the Lytic Phase of the Infection...................................................................................................................... .61 The -83CRE Recombinant Displays Impa ired Replication and Spread in the Nervous System of the Mouse.....................................................................................62 The -83CRE Virus Contains a Second Site Mutation.....................................................64 Mutation of the -83CRE Results in Wild-t ype HSV RNA Levels in RS Cells during Acute Infection.............................................................................................................64 The -83CRE Virus Expresses LAT during Latency........................................................65 The -83CRE Virus LAT Promoter Region’ s H3 K9, K14 Acetylation Levels Are Similar to Those of ICP0 during Latency....................................................................65 The -83CRE Virus Reactivates from Latenc y in the Rabbit with Similar Efficiency as Wild-type 17 syn +....................................................................................................66 Discussion..................................................................................................................... ..........66 Mutation of the -83CRE Is Unfavorable for Recombination..........................................66 The -83CRE Recombinant Contains a Second Site Mutation that Contributes to the Avirulence Phenotype..................................................................................................68 Cell-Specific Factors May Interact with the -83CRE Site to Convey Neuronal Tropism to HSV-1........................................................................................................70 Mutation of the -83CRE Site Does Not Affect Latency and Reactivation......................71 The Wild-Type -83CRE May Control LAT during the Acute Infection.........................73

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6 4 OVERALL DISCUSSION.....................................................................................................84 The LAT and Chromatin........................................................................................................84 LAT Regulation through Promoter Function.........................................................................86 A Model for Reactivation....................................................................................................... 87 APPENDIX REAL-TIME PCR PRIMER/PROBE SEQUENCES...........................................90 LIST OF REFERENCES............................................................................................................. ..91 BIOGRAPHICAL SKETCH.......................................................................................................101

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7 LIST OF TABLES Table page 3-1 Survival of mice infected via the footpad with either 17 syn + or -83CRE.........................74 3-2 Survival of mice inoculated intracranially with either 17 syn + or -83CRE........................74 3-3 Slit lamp examination (SLE) scores of rabb it corneas at 3, 5, or 7 days post-infection....74 3-4 Reactivation of rabbits post-epinephrine induction...........................................................74 A-1 Real-time PCR primer/probes used...................................................................................90

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8 LIST OF FIGURES Figure page 1-1 Diagram of the HSV-1 virion............................................................................................29 1-2 Reactivation critical region ( rcr ).......................................................................................29 1-3 Mutants in the reactiv ation critical region.........................................................................30 1-4 Elements of the core LAT promoter..................................................................................30 2-1 Diagram of the chromatin profile of the latent HSV-1 genome in the mouse...................40 2-2 LAT region histone H3 K4 dimethylation status of latently-i nfected rabbits for 17 syn + or 17 Pst...............................................................................................................40 2-3 Dimethyl H3 K4 status during ep inephrine-induced reactivation of 17 syn +....................41 2-4 Dimethyl H3 K4 status during ep inephrine-induced reactivation of 17 Pst.....................42 2-5 Ratios of average relative H3 K4 dimethylation of 17 Pst to those of 17 syn + for epinephrine-induction in rabbits........................................................................................43 3-1 Diagram of PCR primer locations used in verification of -83CRE mutant virus..............75 3-2 Analysis of PCR products amplif ied from dot blot purification........................................75 3-3 Multi-step viral growth curves (m.o.i. of 0.01) for 17 syn + or -83CRE on RS cells, Neuro-2A cells, and L7 cells.............................................................................................76 3-4 Single-step viral growth curves (m.o.i. of 5) for 17 syn + or -83CRE on RS cells and Neuro-2A cells................................................................................................................. ..77 3-5 Percent survival over the c ourse of acute infection of mice infected with either 17 syn + or -83CRE at 500 pfu, 5,000 pfu, or 50,000 pfu...................................................78 3-6 Relative viral genomes for 8 hours, 2 days or 4 days p.i. of mice infected via the footpad with either 17 syn + or -83CRE..............................................................................79 3-7 Percent survival of mice infected with 500 pfu, 5,000 pfu, or 50,000 pfu of 17syn+, -83CRE, or F8-1 rescuant virus.........................................................................................80 3-8 Multi-step growth curve (m.o.i. of 0.001) on RS cells for 17 syn +, -83CRE, and F8-1....81 3-9 Relative RNA transcript levels durin g acute infection of RS cells with 17 syn + or -83CRE recombinant.........................................................................................................82

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9 3-10 LAT expression during latency in mouse DRG infected with the -83CRE mutant virus.......................................................................................................................... ..........83 3-11 The LAT promoter of the -83CRE mutant virus is decreased in histone H3 K9, K14 acetylation relative to wild-type.........................................................................................83

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10 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 REGULATION OF THE HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) LATENCYASSOCIATED TRANSCRIPT (LAT) By Nicole V. Giordani August 2007 Chair: David C. Bloom Major: Medical Scienc es—Immunology and Microbiology Herpes simplex virus type 1 (HSV-1) establis hes latency in neurons until stimulated by stress to reactivate. The latency-associated tr anscript (LAT) region is the only portion of the latent genome that is actively transcribed dur ing latency. While the LAT region is known to facilitate reactivation, the exact mechanism involved is not known. The overall focus of this dissertation was to investigate elements in the LAT promoter that respond to stress and alter transcription in this regulatory region. Here, the chromatin profile of a reactivation-negative HSV-1 LAT promoter mutant, 17 Pst, was assessed prior to and following adre nergic induction of reac tivation in the rabbit ocular model. In contrast to the la tent chromatin profile of wild-type 17 syn +, 17 Pst showed increased enrichment of dimethyl H3 K4 just upstream of the deleted region, suggesting that deletion of the LAT promoter causes a loss of a regulatory element required for initiation of reactivation. Post-induction of reactivation, no apparent chromatin remodeling occurred for either virus during early times (1, 2, 4 hours). To investigate a possible regulator of th e LAT promoter, a cAMP response element, located 83 nucleotides upstream of the LAT transc riptional start site (-83CRE) was examined. A recombinant virus with mutation of the -83CRE wa s created and analyzed for alterations to the

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11 acute, latent, and reactivation phases of the vira l lifecycle. The -83CRE recombinant was found to have a second site mutation somewhere in th e genome. During acute infection, the -83CRE recombinant displayed attenuated virulence in mi ce when infected via the footpad, and in cell culture the virus showed increased replication in epithelial cells and decreased replication in neuronal cells, suggesting that th e mutations in the -83CRE recombinant play a role in viral replication. The -83CRE recombinant virus wa s also found to establis h latency, express LAT, and reactivate from latently-infected rabbits. These results indicate that th e LAT promoter contains elements that regulate transcription at the level of chromatin, which ma y play a key role in facilitati ng stress-induced reactivation. In addition, data from analyses of the -83CRE r ecombinant suggest that a compensatory mutation generated during viral construction plays a role in virulence and may interact with the -83CRE site to regulate lytic infecti on. Overall, this study invokes a model where the LAT promoter acts as complex regulatory switch that modulates ge ne expression in a tissue-specific manner both during the acute and latent pe riods of HSV-1 infection.

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12 CHAPTER 1 INTRODUCTION Basic Overview The virus family Herpesviridae is composed of a number of double-stranded DNA (dsDNA) viruses that share patter ns of gene expression and the ab ility to undergo latency, a state in which the viral genome is virtually shut down until reactivated (reviewed in Wagner and Bloom, 1997). There are herpesviruses that infe ct a wide range of hosts, from mollusks to mammals. In humans there are eight known herp esviruses, with different primary sites of infection and cell types in which they can become latent. Herpes Simplex Virus type-1 (HSV-1) is a member of the -herpesvirus subfamily, a group whose members—HSV-1, HSV-2, and Varicella-Zoster Virus—have a tropism fo r neuronal cells during latency. HSV-1 is very common in the population, with approximately 60-90% seropositivity in the adult U.S. population (Smith and Robinson, 2002). While the virus usually only causes painful, inconvenient cold sores, it can also cause herpes keratitis, the leading cause of infectious blindness in the U.S. (reviewed in Biswas and Rouse, 2005), and in rare instances, can cause encephalitis and even death (reviewed in Higgins et al., 1993). There is no cure for HSV-1, and while drugs affecting specific vira l targets during the periods of active replication (i.e., aciclovir interferes with HSV-1 DNA replication) can li mit productive infection, they are not effective against the latent virus. Thus, by studying latency and reactivation, we can better our understanding of HSV-1, which can eventually l ead to better treatment options and possibly yield a way to prevent reactivation. Productive Infection An infectious herpes virion consists of a glycoprotein-spiked envelope surrounding the amorphous tegument, an icosahedral capsid, and th e genome-containing core (Figure 1-1). The

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13 HSV-1 genome is large when compared to other viruses (approximately 152 kilobases (kb)) and encodes approximately 80 gene products during productive infection of the mucosal epithelium, the usual site of entry. The expression of gene s is temporally ordered, with immediate-early genes being made before early genes which are made before late genes. Virions can also enter sensory neurons, where the virus can enter late ncy as a nucleosome-asso ciated, nonreplicating episome (Rock and Fraser, 1985; Deshmane and Fr aser, 1989) until stressing stimuli induce the viral genome to reactivate from its quiescent state. At this time the virus can be transported by anterograde axonal transport to once again cause productive infec tion at the epithelial surface. The HSV-1 virion first enters its host cell through glycoprotein–receptor fusion, leaving some tegument proteins, including viral host s hutoff (vhs, which degrad es cellular and viral RNA), in the cytoplasm, while the nucleocapsid tr avels to the nucleus. At the nuclear pore, the viral DNA is released into the nucleus. Accompanying the viral genome into the nucleus is the tegument protein, VP16, which interacts with cellu lar factors to enhance transcription of the virus’s immediate early genes. Once the viral genome is in the host cell nucle us, the lytic cascade of transcription can commence. Each class of genes must be transcribe d in order for transcripti on of the next class to begin. The first group of genes made is the immedi ate-early (IE or alpha) genes. There are five of these genes (ICP4, ICP0, ICP27, ICP22, and ICP 47), and this class provides the gene products necessary for the expression of the next gene class, the early (E or beta) genes. The early genes are crucial for replication a nd include polymerase, proteins for DNA and ORI binding, and the helicase/primase complex. Expression of this class is down-regulated after the start of replication of the viral genome. The final group of genes, the late (L or gamma) genes, provides the more than 30 gene products coding for structural components of the virion.

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14 Once all classes of gene products have been made, the viral capsid assembles in the nucleus and buds through the inner nuclear me mbrane, acquiring the tegument layer and an envelope. The virus particle is de-enveloped at the outer nuclear membrane and reacquires an envelope at the Golgi apparatus. Once the mature virion is made, virions are spread by cell-tocell fusion, or they are released from the cell by exocytosis to ini tiate a second round of infection. Latency and Reactivation Animal Models Studies performed in cell culture have provi ded much information about the biology of HSV-1, particularly molecular details of gene regulation during the acut e infection. However, due to the virus’s ability to unde rgo latency in neurons, studies in cell culture have been limited. While several groups have managed to induce a shut-down state of the viral genome in cell culture (O’Neill et al, 1972; Preston et al, 1991 ; Moriya et al, 1994), none have been shown to sufficiently mimic in vivo latency, so the most relevant means to study HSV-1 latency and reactivation is through th e use of animal models. Both the mouse and the rabbit have been invaluable to the advancement of the understand ing the latent phase of HSV-1 infection. Mouse footpad model. There are several models of la tency and reactivation that employ the mouse. Once the mouse is infected through the eye, footpad or ot her route of epithelial inoculation, the virus will establis h a latent infection within the se nsory ganglia that enervate the region of primary infection. Reactivation is mo st often induced by hyperthermia or explant of ganglia. Clinical reactivation or virus shedding at the site of initial inoculation does not reproducibly occur, thus placing a limitation on the relevance of the mouse models to human infection. However, mouse systems are more rele vant than cell culture, are cost-efficient, and

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15 while reactivation occurs in an in vitro manner, the mouse provides useful insight into the molecular events related to latency and reactivation. For the purposes of this dissertation, of the existing mouse models, only the mouse footpad model has been used for the experiments herein, and therefore, this will be the only mouse model for latency and reactivation that is discussed; this is not intended to trivialize the other murine systems but only to provide the background for later discussion. In the mouse footpad model, the keratinized epit helia of the rear footpa ds are first softened by injection of saline. The footpad is then abra ded with an emery board, allowing a direct route for viral entry into the anesthetized animal. The virus replicates in the footpad and then enters the peripheral nervous system, trav eling to the dorsal root ganglia (DRG), the spinal cord and even the brain. If inoculated w ith a virulent strain of HSV-1, mice succumb to viral encephalitis at approximately day 7 to 10 post-infection. In surviving mice the virus becomes latent in neurons of the DRG, and after 21 to 28 days, there is no longer evidence of acute infection (lytic viral transcription has ceased, th e LAT is expressed, and infectious virus is no longer present). At this time the DRG can be dissected from the m ouse, and the latent state can be assessed or the DRG can be explanted to supplemented media for a given length of time, in order to examine molecular events that occur in response to stre ss. Additionally, the late ntly-infected DRG can be co-cultivated on a cell monolayer to analyze the kinetics or e fficiency of explant-induced reactivation. While these methods are not cons idered to necessarily mirror in vivo or clinical reactivation, DRG explant still pr ovides useful information and can serve as a starting point for investigation of this process.

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16 Rabbit eye model. In human HSV-1 infection, latency occurs in the trigeminal ganglia (TG), and reactivation occurs at the site of primary infection, commonly the mucosal epithelia. This is similar to what is observed in the ra bbit eye model—rabbits are infected through the corneas, latency occurs in the TG, and upon reactiva tion infectious virus can be recovered at the site of initial infec tion. In addition to spontaneous r eactivation, it is po ssible to induce reactivation in latently-infected ra bbits. Iontophoresis of epinephr ine via a direct current to the eye has been shown to reliably induce viral shedding from late ntly-infected rabbits at high frequencies (Kwon et al., 1981). Because epine phrine is a hormone released in response to stress, the use of it seems quite relevant. In fact, when rabbits are administered propanolol, a blocker of the -adrenergic receptors that bind epinephrine, there is a significant decrease in the levels of spontaneous reactivation (Kaufman et al., 1996). At the present time, the rabbit eye model is arguably the most relevant model for reactivati on studies. The Latency-Associated Transcript (LAT) When HSV-1 becomes latent in a neuron, there is an overall shutdown of the genome, with the exception of the latency-associ ated transcript (LAT) region (S tevens et al., 1987). The LAT is approximately 8.3–8.5-kb in length (Dobson et al., 1989; Mitchell et al., 1990), and the primary transcript can be spliced to yield 2.0 and 1.5-kb species (Wagner et al., 1988; Wechsler et al., 1988). The 2.0-kb LAT has been demonstrated to be a stable intron (Farrell et al., 1991), with a half-life of almost 24 hours in cell culture (Thomas et al., 2002), while the smaller 1.5-kb appears to be a smaller splice product of the 2.0-kb intron produced only in a subpopulation of neurons that express the LAT. Furthermore, studies performed with a transgenic mouse containing the LAT promoter through the 2.0-kb intron demonstrated that while the transgene was expressed in various tissue types, high leve ls of 2.0-kb intron accumulation occurred only in

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17 sensory ganglia and appeared to be differentially spliced in thos e tissues (Gussow et al., 2006). While it seems likely that the 2.0-kb LAT intron is important to the virus when in sensory ganglia, no precise function has been ascribed to the LAT splice product s, and the 2.0-kb intron is dispensable for normal reac tivation (Jarman et al., 2002). The LAT during lytic infection While the LAT is the prominent transcriptional unit during latency, LAT is also detectable in murine TG during lytic infection (Spivack and Fr aser, 1988) and is expressed as a late gene in cell culture (reviewed in Wagner et al., 1995). In addition, the LA T promoter is active in both neuronal and non-neuronal mouse tissue during the lytic phase of infection (Jarman et al., 1999), suggesting that simply expressing LAT is not enough to cause latency. Finally, one study demonstrated that in acutely-infected murine ganglia, the virus may simultaneously follow productive and latent pathways, the former show ing expression of lytic genes and decreased LAT levels and the latter disp laying almost no viral transcript ion except for the LAT (Margolis et al., 1992). The study also demonstrated that the two pathways occur in two different subsets of neurons, which likely drives the outcome of the infection into being productive or latent (Margolis et al., 1992). The LAT during latency and reactivation The exact role of the primary LAT in latenc y and reactivation is st ill unknown. Promoter deletion mutants that do not express the LAT re tain wild-type levels of establishment and maintenance of latency (Ho and Mocarski, 1989; Ja vier et al., 1988; Sedara ti et al., 1989; Steiner et al., 1989), arguing against a cri tical role for the LAT in those functions. In contrast, however, establishment of latency by a 1.9-kb deletion muta nt of the LAT promoter through the 5’ exon was reduced by approximately 75% relative to the parental and re scued virus; a significantly higher number of neurons in the TG were destro yed by the mutant than by the wild-type virus,

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18 suggesting a role for the LAT in neuronal surv ival (Thompson and Sawtell, 2001). Neuronal survival and anti-apoptotic activities have been at tributed to the LAT in several studies (Perng et al., 2000; Inman et al., 2001; Ahme d et al., 2002; Jin et al., 20 03), and furthermore, a link between spontaneous reactivation and anti-apopto tic activity has been suggested (Jin et al., 2003). It has also been recently suggested th at the LAT 5’ exon region encodes a microRNA that regulates apoptosis, in order to permit reactiv ation of the virus, alt hough the functionality of this microRNA has yet to be demonstrated in th e context of an HSV-1 infection (Gupta et al., 2006). A clear role for the LAT appears to be in re activation. Promoter deletion mutants that do not express LAT display inefficient in vivo reactivation when assessed in the rabbit (Hill et al., 1990; Bloom et al., 1994; Perng et al., 1994). Add itionally, through analysis of deletion mutants, it was demonstrated initially that the region ne cessary for reactivation (reactivation critical region, rcr ) lies in the first 1.5-kb of th e LAT (Bloom et al., 1996; Perng et al., 1996). This region was further mapped to the first 699 bp of the LAT, after mutants containing 2.0-kb intron deletions displayed wild-type levels of induced reactivation (Jarman et al., 2002). Thus, the region of the LAT that is imperative for nor mal reactivation extends from the core LAT promoter through the 5’ exon (Figure 1-2). Additi onally, this region has al so been demonstrated to possess both enhancer and long-term expressi on functions that allow the LAT promoter to remain highly active throughout latency (Lokens gard et al., 1997; Berthomme et al., 2001). Several studies have been performed in order to further map the rcr These have involved the analysis of mutants with s ubdeletions in the 5’ exon region, a nd several of interest will be discussed here. Mutant 17 Sty contains a 370-bp deletion of mo st of the 5’ exon (Figure 1-3). This mutant generates the 2.0-kb intron, has normal replication ki netics, and displays a normal

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19 level of recovery when latently-infected mice TG are co-cultivated (Maggioncalda et al., 1994). In contrast, 17 Sty shows an approximately 40% reducti on in epinephrine-induced reactivation of the rabbit (Hill et al., 1996). This demonstrat es the importance in validating work performed in the in vitro mouse model in the more in vivo rabbit eye model. Strangely, in contrast to the reduction in induced reactivation of rabbits by the deletion mutant spontaneous reactivation does not appear to be altered from wild-type levels wh en that region is dele ted (Perng et al., 1996b). This suggests that there may be more than one pathway for reactivation—one that is stressresponsive and dependent on some element within th e 5’ exon and one that occurs spontaneously and does not require the region. To further address the role of the 5’ exon region in reac tivation, mutant 17 348 was studied. This mutant contains a 348-bp deletion that is loca ted 217-bp downstream of the LAT transcriptional start si te (Figure 1-3). 17 348 expresses LAT and establis hes a latent infection in rabbits at a level similar to that of wild-type, but like 17 Sty, this mutant di splays a significantly decreased level of epinephrineinduced reactivation (Bloom et al., 1996). Surprisingly, when mutants containing overlapping subd eletions of the 348-bp region (Fi gure 1-3) are tested in the rabbit, levels of induced reactivation are ne ar that of wild-type (Bloom et al., 1996; Bhattacharjee et al., 2003). This suggests that there are multiple cis elements in the region that play a role in reactivation and se veral must be deleted in order to detectably alte r reactivation. The region is clearly quite complex, and there may be different means for the virus to reactivate, possibly requiring interplay between va rious components. LAT Promoter and LAT Transcription Wild-type expression of the LAT—and therefor e, wild-type reactivation in the rabbit—is dependent upon the presence of a 202-bp core promot er (Dobson et al., 1989). In fact, when the core LAT promoter is deleted from wild-type 17 syn + to generate 17 Pst, epinephrine-induced

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20 rabbit reactivation (recovery of in fectious virus) is significantly decreased, from approximately 79% of 17 syn +-infected rabbits positive for infe ctious virus to about 13.5% of 17 Pst-infected rabbits positive (Bloom et al., 1994). The differen ce is not as dramatic when the DRG or TG are explanted from the mouse to cell culture. Approximately 50% of 17 Pst-infected ganglia reactivate (cause visible cytopa thic effect when explanted to a cell monolayer) while nearly 100% of 17 syn +-infected ganglia reactivate (Devi-Rao et al., 1994). This suggests that the presence of the core LAT promoter may have a slight effect on explant-reactivation but appears to be more important for epinephrine-induced reac tivation in the rabbit. While this is likely due to a function of the LAT, it may also be due to a cis -acting element contained within the region. Within the 202-bp of the core LAT promoter ar e several transcripti on factor binding sites that appear to be important for the full functi on of the core LAT promoter. Among the identified sites are a TATA box (Dobson et al., 1989), two cAMP response elem ents (CREs) (Leib et al., 1991; Kenny et al., 1994), and binding sites fo r the upstream stimulatory factor (USF) (Zwaagstra et al., 1991; Kenny et al., 1997) and ICP4 (Batchelor et al., 1994) (Figure 1-4). The TATA box is necessary for normal f unction of the LAT promoter. When chloramphenicol acetyltransferase (CAT) assays were performed using a plasmid in which the TATA box was mutated or delete d, a reduction in promoter activ ity was observed relative to wild-type (Rader et al., 1993; Ackland-Berglund et al., 1995). In vitro transcription assays also demonstrated that the TATA box is required for full LAT expression (Soares et al., 1996). In addition, it was demonstrated that co-cultivation of cells with murine TG latently-infected with a TATA box mutant virus yielded normal levels of reactivation via this method, even though the mutant displayed significantly decreased levels of LAT expression, as assayed by in situ

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21 hybridization (Rader et al., 1993). In other words, the ability of HSV-1 to express LAT does not necessarily correlate with the ability to reactivate from explanted murine ganglia. The CRE identified in the LAT promoter locat ed approximately 43 base pairs upstream of the LAT transcriptional start site (referred to here as -43CRE) (Leib et al., 1991) was demonstrated to bind the CRE-binding type 1 (C REB-1) protein (Mill house et al., 1998), a known stress-responsive transcri ptional activator. When the -43CRE was deleted and its promoter activity was assayed by CAT assay, a decrea se of threeto four-fold relative to wildtype was observed (AcklandBerglund et al., 1995), even though LAT expression was not affected when assayed by ribonuc lease protection assay or by in situ hybridization (Rader et al., 1993; Ackland-Berglund et al., 1995). It was therefore suggested that the -43CRE has an inducible rather than basal activity in the cont ext of the LAT promoter (Ackland-Berglund et al., 1995). Interestingly, the spacing of the -43CRE s ite relative to the TATA box appears to play a role in activity. Insertion of 10 nucleotides between the CRE and the TATA box of the LAT promoter resulted in 2-3 fold more CAT activity compared to that of the wild-type promoter, while removal of 5 nucleotides decreased activit y by 6-8 fold relative to wild-type; since the 10 bp spacing reflects a turn of the DNA helix, this suggests a requirement for interaction between factors binding to the two elements (Ackland-Berglund et al., 1995). Because the cAMP response pathway is activat ed in response to binding of epinephrine to cell receptors, it seems likely that the CREs in HSV1 play some role in reactivation. However, when the -43CRE site deletion mutant was tested in the rabbit, epinephr ine-induced reactivation was intermediate between wild-type strain 17 syn + and promoter-deletion mutant 17 Pst (Bloom et al., 1997), suggesting that the -43CRE was not the only factor aff ecting reactivation. Interestingly, LAT expression dur ing latency was similar between the -43CRE mutant virus and

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22 wild-type (Bloom et al., 1997). This may indicat e that the ability to express the LAT does not necessarily correlate with reactivation, but in stead, elements within the LAT promoter may interact with cellula r and viral factors to yield wild -type levels of reactivation. A second CRE was identified between nucleo tides -75 to -83 relative to the LAT transcriptional start site (-83 CRE) (Kenny et al., 1994). This 83CRE was demonstrated to bind a repressive form of CRE bi nding (CREB) protein, CREB-2 (Mil lhouse et al., 1998), suggesting that this site may play a role in transcri ptional repression of LAT. Other findings and speculations on the role of the -83CRE will be discussed further in Chapter 3. In addition to the CREs, a site capable of binding USF is present in the LAT promoter (Zwaagstra et al., 1991; Kenny et al., 1997). USF binds to the Ebox of a promoter and interacts with transcriptional machinery as well as with chromatin remodeling proteins (reviewed in Corre and Galibert, 2005). In bovine leukemia virus (BLV ), gene expression of the 5’ long terminal repeat appears to be regulate d by the exclusion of CREB from a CRE that overlaps an E-box; mutation of the E-boxes appears to increase binding of CREB complexes to the CRE and also increases gene expression (Calomme et al., 2004). Through electrophoretic mobility shift (EMS) assay, it was dem onstrated that the HSV-1 LAT promoter E-box can bind eith er of the two forms, USF-1 and USF-2 (Kenny et al., 1997). When the -43CRE and E-box of the HSV-1 LAT promoter were mutated simultaneously and examined in an in vitro transcription assay, transc ription levels appeared to decrease more than when either element was mutated alone, suggesti ng an interplay between the two (Soares et al., 1996). Neither the potentia l interactions between the E-box and the -83CRE, nor the effects of these elements on reactivation or LAT transc ription in vivo have been investigated.

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23 The LAT promoter, in addition to various transc ription factor binding s ites, also has a site to which ICP4 can bind (Batchelor et al., 1994). When this ICP4-binding site is mutated to abrogate binding, in vitro expression of the promoter occurs at aberrant times, displaying early gene expression, as opposed to the usual late ge ne expression kinetics (Rivera-Gonzalez et al., 1994). In other words, it appears that the ICP4 site may act to control LAT expression at inappropriate times, such as during produc tive infection and reactivation. In summary, the core LAT promoter possesses several binding sites that appear to play some role in regulation of the LAT. It is possibl e that some of these func tion in certain cell types and at certain times during the infection to co ntrol the region. Additionally, various studies suggest interplay between different elements, i ndicating that this region is both complex and important. Eukaryotic Epigenetic and Transcriptional Regulation HSV-1 gene regulation shares similarities with that of eukaryotes, including an association with nucleosomes during latency (Deshmane and Fraser, 1989) and various eukaryotic transcription factor binding site s throughout the genome. While th ere are other similarities, for the sake of brevity, only the rele vant aspects of epigenetics to HSV-1 transcriptional regulation will be discussed here. Epigenetic Regulation Epigenetics can be defined as a modification in gene expression or cellular phenotype that does not change the actual DNA (reviewed in Gol dberg et al., 2007). More specifically, protein interactions with DNA are capable of causing changes in gene expression. In order for DNA to readily fit into cells, th e genome is compacted into chromatin fibers, which is generally grouped into two different classes, euchromatin and he terochromatin. Euchromatin is a more relaxed structure, which allows access to the DNA by a range of transcription fact ors and other various

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24 proteins, while heterochromatin is condensed chromatin with DNA that is inaccessible to transcription factors and is ther efore transcriptionally inactive. The basic unit of chromatin, both euchromatin and heterochromatin, is the nucleosom e, which consists of 147 bp of DNA wrapped around an octamer of four core histones (H2A H2B, H3, and H4) (reviewed in Kouzarides, 2007). Interactions of nucleosomes and DNA can be altered by three mechanisms: posttranslational modifications (PTMs), replacemen t with histone protein variants, and ATPdependent chromatin remodeling (reviewed in Bernstein and Hake, 2006). However, since PTMs are more relevant than the others to the wo rk in this dissertation, they will be discussed here, and while a variety of PTMs have been char acterized, only those that are most pertinent to the work in this dissertation will be reviewed. The addition of PTMs to the Nor C-terminal tails of histones can al ter the transcriptional permissivity of the nucleosomes. Histone H3 ac etylation of lysine residues 9 and 14 (K9, K14) and dimethylation of lysine residue 4 (K4) are bo th traditionally associated with regions of active transcription (reviewed in Li et al., 2007). A yeast microarray study found that while histone H3 K4 dimethylation was not globally correlated with promoter regi ons of transcri ptionally active genes, there was a statistically significant asso ciation between the modi fication and the coding regions of active genes (Bernstein et al., 2002). In contrast, the study also demonstrated that H3 K9, K14 acetylation was associated with the promot ers of active genes, as well as within coding regions, although to a slightly le sser extent. A separate study performed using genome scanning of two human cell lines revealed that of 57 active genes analyzed, 58% displayed enrichment in histone H3 K4 dimethylation and H3 K9, K14 a cetylation within 500 bp of transcriptional start sites, while 28% were farther down in the coding region (Liang et al., 2004). Thus, while there may be subtle distinctions between where the PTMs are associated, it is commonly accepted that

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25 histone H3 K9, K14 acetylation a nd histone H3 K4 dimethylation are markers of transcriptional activity. Chromatin studies performe d on HSV-1 have focused mainly on active marks of chromatin. During lytic infection of cell cultu re, it was shown that while the DNA is in a partially nucleosomal state, association of the active histone mark, acetylated H3 K9, with viral DNA occurs by one hour post-infection (p.i.) for ICP0, thymidine kinase, and VP16 (Kent et al., 2004). However, contrary to that study, Herrera and Triezenberg (2004) de monstrated that very little histone H3 (nonacetylated or acetylated) is present at the IE ge ne promoters examined (ICP0, ICP4, ICP27) during early lytic infection (2 h.p.i.), while the thymidine kinase, VP16 and glycoprotein C promoters are associated with a cetyl-H3 K9, K14. One interesting difference in these lytic infection experiments is that the form er, in which acetylated H3 K9 was associated with viral genes of all classes by 1 h.p.i., was performed in the neuronal SY5Y cell line, while the latter was performed in the more epithelial-like HeLa cells. Differential chromatin patterns may indicate that the chromatin conformation, incl uding the association with histone H3, of the HSV-1 genome differs between cell types, possibl y impacting establishment of latency. The ability of HSV-1 to exist as a represse d episome during latency suggests that viral repression may occur at the level of chromatin. In fact, one study suggested that expression of the LAT may cause increased levels of H3 K9 dimethylation and decreased levels of H3 K4 dimethylation of lytic gene promoters during mu rine infection (Wang et al., 2005). When the LAT region is examined during latency, increased levels of H3 K9, K14 acetylation are observed for the LAT promoter and 5’exon/enhancer regi ons relative to lytic ge nes (Kubat et al., 2004a; Kubat et al., 2004b). Further, explant reactivation of murine DRG appears to induce dramatic changes at early times in both the LAT region’s and ICP0’s transcriptio nal permissiveness as

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26 assayed by acetyl-H3 K9, K14, whereby the LAT re gion seems to decrease in acetylation before ICP0 can begin to increase (Amelio et al., 2006 ). Thus, the dynamic regulation of chromatin modifications appears to also impact the HS V-1 genome’s transcriptional permissiveness at various stages during infection. Promoter Elements and Transcriptional Regulation In most eukaryotic cells, an active gene prom oter contains a nucleosome-free region (NFR) approximately 150 bp in size surrounding the core promoter (reviewed in Heintzman and Ren, 2006). After the region is hyperacetylated, ch romatin remodeling oc curs and histone-DNA contacts are lost or nucleosome-unfolding ta kes place (Boeger et al., 2003; Reinke and Hrz, 2003). This allows for binding and stabilization of the transcriptional machinery to the promoter (reviewed in Heintzman and Ren, 2006). While there are three types of RNA polymera ses—I, II, and III—RNA polymerase II (Pol II) is responsible for transcription of mRNA a nd other regulatory RNAs and will therefore be discussed here. Transcription can initiate once Pol II is recruited to a gene’s core promoter, which surrounds the transcripti onal start site and encompasse s 70–80 surrounding base pairs that are recognized by the transcriptional machinery, but the surrounding sequences may be part of the proximal promoter, conveying tissue-specifici ty or acting as a tran scriptional enhancer (reviewed in Heintzman and Ren, 2006). There is much variability in th e factors that are bound to a specific promoter, but promoters generally function in a similar mann er. First, chromatin remodeling allows Pol II and other transcription factors to gain access to the promoter; this preinitiation complex (PIC), once properly positioned, melts the 11–15 bp of DNA around the transcriptional start site for correct interaction of the Pol II with the DNA and then begins transcription (reviewed in Heintzman and Ren, 2006).

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27 Binding factor recruitment is es sential for transcriptional activ ation, but proteins that bind to a promoter may instead function to repress th e gene. While active tran scriptional repressors target a gene at the level of chromatin, passive repressors can compete with transcriptional activators for binding, bind to activat ors as inactive heterodimers to inhibit transcription, or bind to coactivators to prevent activation of transcript ion factors (reviewed in Thiel et al., 2004). One example of a transcriptional repressor is the in ducible cAMP early repressor (ICER), which has been suggested to play a role in HSV-1 reac tivation through repression of LAT (Colgin et al., 2001). ICER is expressed from the same locus as CREB, a transcriptional activator, but is transcribed from an intronic promoter and does not contain an activation domain (reviewed in Mayr and Montminy, 2001). ICER levels peak at 2–6 hours after cAMP stimulation, and ICER prevents CREB from binding to CRE-containing pr omoters, including the promoter which drives ICER’s own transcriptio n (reviewed in Mioduszewska et al., 2003). Numerous other examples of transcriptional repressors exist in eukaryotes and in c onjunction with transcriptional activators, can allow promoters to functi on as switches for transcription. Summary Many questions still remain regarding the HSV1 LAT’s role in regu lation of the viral lifecycle. Numerous experiments have demonstrat ed that one major func tion appears to be in reactivation, since the LA T promoter and the 5’ exon are critical to wild-type r eactivation. Other activities have also be en ascribed to the region, and a pi cture is emerging in which LAT may play a larger regulatory role, with some of th e various phenotypes attributed to LAT mutants being secondary effects of this regulatory function. Clearly, understanding how LAT is regulated at the level of transc ription and in different cell type s may provide insight into its larger role on HSV biology. Experiments detail ed in this dissertation were aimed to address some of the questions regarding LAT regulatio n—specifically, (1) what is the importance of

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28 histone tail modifications in the ability to reactivate from latency, and (2) does a stressresponsive CRE in the LAT promoter play a regula tory role in reactiva tion? The answers to these questions will hopefully provide some indication of the complex regulation of the HSV-1 LAT region, as well as open up avenues for future work.

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29 Figure 1-1. Diagram of the HSV-1 virion. Th e envelope, tegument, capsid, and core are indicated. Figure 1-2. Reactivation critical region ( rcr ). The minimal region, mapped by various subdeletions, that is necessary for wild-type in vivo reactivation in response to stress is designated by the blue bar. The core LAT promoter (LAP1), shown in yellow, is defined here as the promoter region encompassed by the two Pst I restriction enzyme sites. The LAT enhancer, extending through th e 5’ exon, is shown in red. Note that this is not to scale.

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30 Figure 1-3. Mutants in the reac tivation critical region. Normal reactivation, shown in black, refers to the ability to reactiv ate at wild-type levels in the in vivo rabbit reactivation model. Red bars indica te decreased reactivation. Figure 1-4. Elements of the core LAT promoter (defined as the region encompassed by the two Pst I restriction enzyme sites).

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31 CHAPTER 2 CHROMATIN CONFORMATION OF THE LATENT HSV-1 GENOME IN RABBITS Objective Preliminary work in the mouse model demonstr ated that the HSV-1 genome is associated with specific histone modifications during late ncy and that there are changes in chromatin permissiveness that occur during explant induced reactivation. The goals of the studies performed here were to inves tigate patterns of histone modifi cations in the rabbit, a more relevant model for HSV reactivation and to (1) determine if the histone modifications observed during latency in mice are conserved in the rabbit eye model, (2) determine if epinephrineinduced reactivation results in the same pattern of chromatin changes as observed in the mouse during explant, and (3) determine if the chroma tin profile of a reactiv ation-negative mutant differs from wild-type during late ncy and/or reactivation (this coul d suggest that the defect in reactivation is related to inappropriate chromatin configuration affecting promoter accessibility and/or transcriptiona l permissiveness). Background and Previous Findings HSV-1 is maintained as a nucleosome-associat ed episome during latency (Deshmane et al., 1989). This observation suggested that insight into the transcri ptional status and regulatory framework could be determined by analyzing the specific histone m odifications that are associated with the different regions of th e HSV genome. In order to analyze histone modifications of the latent and reactivating HSV-1 genome, the chromatin immunoprecipitation (ChIP) assay has been used. In the ChIP a ssay (reviewed in Kuo and Allis, 1999), histones are crosslinked to DNA using formaldehyde, and these complexes are sonicated into fragments of approximately 500–1000 bp. Histones are immunopr ecipitated with the antibody of choice and then de-crosslinked from the DNA. The freed DNA is then purified and analyzed by PCR.

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32 As discussed in Chapter 1, there are two pr imary animal models used for the study of HSV-1 latency and reactivation, the mouse and the rabbit. The mouse footpad model has enabled much of the work related to epigenetic studies of the HSV-1 genome during latency and has also provided some insight into chromatin remodeling that occurs during early DRG explantinduced reactivation. Using the mouse footpad model, it was determ ined that DNA methylation does not play a role in the repression of the latent HSV-1 genom e (Kubat et al., 2004). Instead, since the latent genome is associated with nucleosomes (Deshm ane et al., 1989), histon e tail modifications appear to provide some indication as to tran scriptional permissivene ss (Kubat et al., 2004). Specifically, during latency the LAT promoter regi on is 2–3.5 fold more enriched in acetylated histone H3 K9, K14, a marker of transcriptional permissiveness, th an the lytic genes, ICP27 and ICP0 (Kubat et al., 2004). Upon further a ssessment of the LAT region—specifically the enhancer located in the 5’ exon—it was observed that the enhancer region is more acetylated (approximately 3.5 fold) than the LAT promoter, while lytic genes exist in a hypoacetylated, or less transcriptionally permissive, state (Kubat et al., 2004). The sa me effect was seen for a LAT promoter deletion virus, which makes no detectable LAT. With this mu tant the LAT enhancer was still hyperacetylated relative to the LAT prom oter, which was still more acetylated than the nearby lytic genes (Kubat et al., 2004 ). In summary, this previous work indicates that the LAT region is maintained in a transc riptionally permissive state independent of LAT transcription, while the lytic gene regions of the HSV-1 genome exist in a le ss transcriptionally permissive state during latency (Figure 2-1). The findings for the latent genome’s chromatin configuration were extended to the mouse explant model, which provides information about the molecular events in reactivation. The

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33 question addressed was whether specific re gions of the HSV genome undergo chromatin remodeling in response to explant-induced stress. If so, this could indicate what key changes occur early in reactivation. La tently-infected DRG were removed to media for times ranging from 0 hours post-explant (h.p.e) to 4 h.p.e. a nd then processed for ChIP. The LAT 5’ exon/enhancer displayed at least a five-fold decr ease in H3 K9, K14 acetylation occurring within the first hour of explant, while the ICP0 promoter exhibited an increase in acetylation between 2 and 3 h.p.e. (Amelio et al., 2006). This study ad ditionally found that there was a dramatic decrease in LAT RNA abundance between 2 and 3 h.p.e. (Amelio et al., 2006). Overall, these findings suggest that there is a remodeling of the LAT region during early explant, whereby both deacetylation of the LAT enhancer and a decrease in LAT levels occur before the ICP0 promoter can become more acetylated. The mouse explant model is limited by allowing only in vitro reactivation studies to be performed. The type of r eactivation obtained through DRG explant is relatively LATindependent, so molecular reactivation occurs re gardless of whether a mutant virus does not transcribe LAT. A more relevant model is the rabbit eye model. Epinephrine-induced reactivation of the rabbit occurs in vivo can produce clinical lesions and shed virus at the eye, and reactivation is more LAT-dependent, in that LAT promoter deletion mutant are severely reduced in reactivation relative to wild-type (Hill et al., 1990; Perng et al., 1994). It is for this reason that many of the studies initially performed using the m ouse footpad model are validated in the rabbit. The experiments described here u tilized the rabbit eye model to determine if the remodeling events observed in the mouse also occur following adrenergic induction of reactivation in the rabbit.

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34 Materials and Methods Rabbit Infections One to 2 kg New Zealand White rabbits were in fected and housed at the Louisiana State University Health Science Center’s Animal Facility. Each rabbit eye received topical proparacaine-HCl anesthesthetic prior to corneal s carification. Rabbits were infected with either 17 syn + or 17 Pst virus inoculum at 50,000 pfu/eye. At days 3, 5, and 7 post-infection (p.i.), the infection of the rabbit eyes was monitored by slit lamp examination for the presence of dendrites on the cornea. After 28 days p.i., rabbits were sacrificed (lat ent time point) or epin ephrine-induced prior to sacrifice (reactivati on time points). If epinephrine iont ophoresis was performed, rabbits were anesthetized with isoflurane, and a solution of 0.015% epinephrine was administered to the rabbit eye for 8 minutes at 0.8 mAmps. At 0, 1, 2, or 4 hours post-induction, rabbits were anesthetized with ketamine/xylazine and euthanized with a lethal dose of sodium pentobarbital. After decapitation, rabbit trigeminal ga nglia (TG) were removed and processed. ChIP Assay ChIP assays were performed at the University of Florida. Rabbit TG were homogenized in 0.5 ml phosphate-buffered saline (PBS) in the presen ce of protease inhibitors (1 g/ml aprotinin, 1 g/ul leupeptin, and 1 mM PMSF). DNA-histone complexes were crosslinked by the addition of 37% formaldehyde to a final concentration of 1%. After th e addition of 0.128 M glycine, the sample was pelleted and washed three times with PBS containing pr otease inhibitors as described above. After the final wash, pellets were resuspended in SDS-lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl), and sonicated (Fisher Sonic Dismembrator 100) to yield fragments of 500-1000 bp (Setting 4, 6 bursts of 40 sec. each). Sonicated samples were precleared with Salmon Sperm DNA/Protein A Ag arose beads (Upstate), and histone-DNA

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35 complexes were immunoprecipitate d overnight with 3.5 g/ml of an ti-acetyl-Histone H3 K9/K14 (Upstate) or 1 g/ml of anti-dimethyl-H3 K4 (Ups tate). Prior to the wash steps, 25% of the sample was removed and retained as the “unbound” fraction. Complexes were washed with Low Salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA 20 mM Tris-HCl, 150 mM NaCl), High Salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), LiCl (0.25 M LiCl, 1% Nonidet P-40, 1% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl), and TE (10 mM Tris-HCl, 1.2 mM EDTA) wash buffers prior to the immune complexes being eluted from the agarose beads with elution buffer (1% SDS, 0.1 M sodium bicarbonate). DNA (bound sample) was de-crosslinked from histones with 10 l/ml 5 M NaCl and then treated with 20mg/ml RNase A and 40 g/ml Proteinase K. DNA (bound and unbound fractions) wa s purified using a QIAquick PCR Purification kit (Qiagen) be fore analysis by Taqman real-time PCR. Taqman Real-Time PCR Analysis Bound and unbound DNA was amplified by real-time PCR using TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Bios ystems) and FAM-labeled TaqMan targetspecific primer/probe. Reactions were run in triplicate in concentrations recommended by the manufacturer. Primer and probe sequences ar e shown in Table A-1. PCR was performed and analyzed using Applied Biosystems 7900HT Se quence Detection Systems. Cycle conditions used were as follows: 50C for 2 min. (1 cycle) 95C for 10 min. (1 cycle), 95C for 15 sec., and 60C for 1 min. (40 cycles). Threshold values used for PCR analysis were set within the linear range of PCR target amplificati on. Average cycle threshold (Ct) values were determined, and the relative quantity was calculate d using a standard curve specific for the primer/probe set of interest.

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36 Results The Chromatin Profile of the Latent HSV Geno me in Rabbits Latently-Infected with WildType HSV-1 Is Similar to that Observed for Latently-Infected Mice When the ChIP assay was performed on rabbit TG that were latently-infected with wildtype 17 syn +, the anti-dimethyl H3 K4 profile revealed an enrichment in th e histone modification for the LAT region relative to the lytic genes ICP0 and ICP27 (Figure 2-2A). This was similar to the findings obtained for the latent ly-infected mouse, in which transcriptional permissiveness of the LAT 5’ exon/enhancer was approximately 3.5 tim es greater than that of the LAT promoter region and almost 40 times greater than that of the lytic genes, ICP0 and ICP27 (Kubat et al., 2004). Thus, it appears that wild -type HSV-1 establishes a similar latent chromatin profile in both the footpad-infect ed mouse and the ocul arly-infected rabbit. Deleting the Core LAT Promoter Results in Increased H3 K4 Dimethylation of the LAT Promoter Region in Latently-Infected Rabbits The chromatin profile of HSV-1 genomes in latently-infected mice, in which the 5’ exon/enhancer is more transcrip tionally permissive than the LAT promoter or nearby lytic genes, is the same for both a wild-type and a promoter-d eletion (LAT-negative) mutant (Kubat et al., 2004). The profile observed in latently infected rabbits was strikingly different. As shown in Figure 2-2B, on average the LAT promoter regi on (amplified by primers approximately 300 bp upstream of the deletion) in the LAT core promoter deletion mutant, 17 Pst, is 8.5 times more enriched in dimethyl H3 K4 than the wild-typ e promoter region. The 5’ exon/enhancer, ICP0, and ICP27 all show similar levels of enrichment between the two viruses. Since the only region with a dramatic difference between 17 syn + and 17 Pst is the LAT promoter, this suggests that the region deleted in 17 Pst contains a cis -element with repressive activity.

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37 Neither 17 syn + nor 17 Pst Display Dynamic Changes in the LAT Region in Response to Epinephrine Induction of Latently-Infected Rabbits Since explant of mouse DRG appeared to indu ce dramatic changes in the chromatin profile at early times post-explant, one would surmise th at iontophoresis of rabbits would induce similar changes. However, iontophoresis of rabbits at 0, 1, 2, and 4 h, resulted in no significant change (Figure 2-3). As shown in Figure 2-4, the LAT promoter region of 17 Pst remains highly enriched in dimethyl-H3 K4 rela tive to the other targets tested for all times examined. These findings suggest that events related to chromatin remodeling might not occur as rapidly in the rabbit as they do in the mouse or that during latent establishment in the rabbit, the LAT promoter plays a different role than in the mouse in establ ishing a chromatin profile that is permissive for normal reactivation to occur. Relative H3 K4 Dimethylation Levels of the LAT Promoter Region in 17 Pst Are Higher than Those of Wild-type for All Times Examined The scale of bound/input (B/U) ratios for the 17 Pst ChIP experiment is much higher than that observed for 17 syn +, as visible by comparing Figures 2-3 and 2-4. The difference can be appreciated by the comparison of the average va lues for the times examined. As shown in Figure 2-5, when the ratios of the average 17 Pst to 17 syn + dimethylation levels are determined, the 5’ exon/enhancer, ICP0 and ICP27 all show ratios of approximately one, indicating similar levels of dimethylation, while the LAT promoter ratios range from approximately 5 to 22-fold more dimethylation in 17 Pst than in 17 syn +. This hyperdimethylati on suggests that the region deleted contains an element that normally represse s H3 K4 dimethylation in the wild-type virus. Discussion Kinetics of Chromatin Remodeling Upon explant of latently-infected murine DRG, a rapid loss of H3 K9, K14 acetylation of the LAT 5’ exon/enhancer is seen within the fi rst hour of explant (Amelio et al., 2006). This

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38 decrease is approximately 5-fold less than what is observed initially at 0 h.p.e. and precedes an increase in acetylation of the ICP0 promoter that occurs between 2 and 3 h.p.e. (Amelio et al., 2006). This timing does not appear to be the same in the rabbit model. When the latent HSV-1 chromatin profile was assessed for rabbit TG infected with wild-typ e virus, the virus displayed a similar latent profile to that of the latently-inf ected mouse. However, when the virus’s chromatin profile was examined after epinephrine-induced reactivation (1, 2, and 4 hours post-epinephrine induction), it did not mirror the findings from th e mouse explant experiment s. In fact, little, if any, change is observed at all with in the first four hours of epine phrine-induction (Figure 2-3). Since the timing of chromatin ch anges does not appear the same as is seen in the mouse, it is possible that there are different mechanisms by which latent HSV-1 can reactivate. The mouse DRG explant model is dependent on a more stress ful reactivation stimulus i.e., the removal of tissue, while the rabbit eye model allows for a more clinically-relevant epinephrine-induced reactivation. A more physiologica lly relevant stimulus might cau se a more gradual change of latent genome configuration. Perhaps at a singlecell level, changes occur quite rapidly, but they do not translate to the level of the tissue until slightly later. For example, since the epinephrine is administered to the eye and reactivation occurs in the ganglia, it might take some time for the epinephrine to reach and stimulate all cells in the ganglia; in explan t-induced reactivation, a uniform stressor (ganglion removal from the animal ) might stimulate cells to reactivate very rapidly. Thus, it remains possible that if rabb it TG are analyzed for chromatin at later time points, changes might be apparent, and these experiments are underway. A Repressive Element in the LAT Promoter When the chromatin profile of the LAT promoter deletion mutant, 17 Pst, is analyzed, there is a striking increase in tr anscriptional permissiveness of th e region slightly upstream of the deleted region, while the remainder of the vira l targets tested show levels of H3 K4

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39 dimethylation that are comparable to wild-type (Figures 2-2). This suggests the presence of a repressive element in the native core LAT promoter that might pr event increased transcriptional permissiveness of the LAT region at inappropria te times, such as during lytic infection. Additionally, since efficient react ivation from latency in the rabbit appears to require LAT expression (Hill et al., 1990; Perng et al., 1994), LAT may play a role in establishing the genome in a configuration compatible with reactivation. It has been proposed by Bloom et al. (1996) that like XIST, a non-coding RNA that silences the inactive X chromosome, the LAT RNA acts in cis to silence the HSV-1 genome dur ing latency. Perhaps the LAT functions differently and is less important in the mouse. Since explant of mouse DRG can induce reactivation of promoterdeletion viruses that would not efficiently reactiv ate in the rabbit, a co rrect chromatin profile may be less crucial for reactivation in the mouse. The 202-bp core LAT promoter contains a number of binding sites for factors that may, in turn, bind various chromatin modifiers. Further i nvestigation of this region should yield valuable insight into the mechanisms of HSV-1 reactivation.

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40 Figure 2-1. Diagram of the chromatin profile of the latent HSV-1 genome in the mouse. The LAT promoter is designated by the light gr ay box, while the LAT enhancer is shown in dark gray. Green bars indicate regions of higher transcriptional permissiveness, while red bars indicate less transc riptionally permissive regions. Figure 2-2. LAT region histone H3 K4 dimethylation status of la tently-infected rabbits for A) wild-type 17 syn +, or B) promoter-deletion mutant 17 Pst. Relative Bound/Unbound, B/U, values are depicted for the LAT prom oter, 5’ exon/enhancer, ICP0, and ICP27.

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41 Figure 2-3. Dimethyl H3 K4 stat us during epinephrine-induced r eactivation at A) 0, B) 1, C) 2, and D) 4 hours post-epinephrin e (h.p.e.) for wild-type 17 syn +. Relative Bound/Unbound, B/U, values are depicted fo r the LAT promoter, 5’ exon/enhancer, ICP0, and ICP27.

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42 Figure 2-4. Dimethyl H3 K4 stat us during epinephrine-induced r eactivation at A) 0, B) 1, C) 2, and D) 4 hours post-epinephrine (h.p.e .) for promoter-deletion mutant, 17 Pst. Relative Bound/Unbound, B/U, values are depicted for the LAT promoter, 5’ exon/enhancer, ICP0, and ICP27.

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43 Figure 2-5. Ratios of average rela tive H3 K4 dimethylation of 17 Pst to those of 17 syn + for epinephrine-induction in rabbits (0, 1, 2, a nd 4 hours post-induction). Ratios for the LAT promoter (L. Pro.), LAT enhancer (5’ exon), ICP0, and ICP27 are shown.

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44 CHAPTER 3 INVESTIGATION OF THE ROLE OF A LA T PROMOTER cAMP RESPONSE ELEMENT (CRE) IN REACTIVATION Objective It has been previously shown that LAT abunda nce transiently decrea ses following explantinduced reactivation, suggesting that regulation of LAT expr ession may be an important component of the switch between latent and productive infection. The LAT promoter contains various elements that may be important regu lators of the LAT region during latency and reactivation. The studies descri bed here were performed to inve stigate the role of one of the LAT promoter’s cAMP response elements (CREs) in reactivation. To do so, a virus was constructed with a site-directed mutation in the CRE and assessed for (1) in vitro replication in cell culture, (2) replication and virulence in the mouse, (3) ab ility to establish latency and express the LAT, (4) chromatin profile during late ncy, and (5) ability to reactivate from latency in the rabbit. Background and Previous Findings Since reactivation of HSV-1 from latenc y is a stress-inducible phenomenon, it seems plausible that the stress-responsive cAMP pathway could play a role in regulation, particularly since the LAT promoter contains two CREs (Lei b et al., 1991; Kenny et al., 1994). The cAMP pathway’s cascade of events is tr iggered upon epinephrine binding to -adrenergic receptors of cells. Once binding occurs, adenylyl cyclase converts ATP to cAMP (Tao and Lipmann, 1969), which can then activate the cat alytic subunit of protein ki nase A (PKA). This subunit translocates to the cell nucleus, where it can phosphorylate members of the cAMP response element binding (CREB) protein family (Montmi ny and Bilezikjian, 1987; Yamamoto et al., 1988). The CREB activator proteins contain a kinase-inducible domain (KID) flanked by glutamine-rich regions at the amino-terminus a nd a basic region/leucine zipper (bZIP) domain at

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45 the carboxy-terminus, which is important in DNA binding and nuclear translocation (Dwarki et al., 1990; Waeber and Habener, 1991; Xing a nd Quinn, 1994). Since th e CREB gene undergoes alternative splicing to yield various family members, truncated vers ions of the protein that lack an activation domain, can cause tran scriptional repression rather than activation (Karpinski et al., 1992; Molina et al., 1993). Once CREB is phosphorylated and activated at a gene’s promoter, it facilitates recruitment of CREB-binding protein (CBP) (Chrivia et al., 1 993), which in turn can promote transcriptional activation (Arias et al., 1994; Kw ok et al., 1994). Although some questions remain as to the exact role of CBP in transcri ptional activation, it ha s been implicated in promoting rapid formation of the preinitiation complex (PIC) to in crease the rate of tran scription and has also been suggested to facilitate r ecruitment of mediator complexes to active sites of transcription (reviewed in Vo and Goodman, 2001). Because CBP is a histone acetyltransferase (HAT), it likely contributes to promoter activ ation through chromatin remodeling (Ogryzko et al., 1996). Genome-wide characterization of CREB binding to various target genes in human tissues revealed that while CREB occupies approximately 4,000 promoters, only a small subset of those genes are actually activated in response to cAMP, which is li kely due to a requirement for coactivator recruitment (Zhang et al., 2005). In other words, CREB binding is constitutive, and regulation is dependent on CBP and possibly, other coactivators. In contrast, one study demonstrated, through the use of chromatin imm unoprecipitation (ChIP) a ssays, that binding is regulated in a cell-specific manner, correlati ng with the potential for gene expression (ChaMolstad et al., 2004). This study also found that histone H3 K4 dimet hylation corresponds to CREB binding, suggesting either that CREB binding to a promoter is regulated in an epigenetic

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46 manner or that CREB binding is indicative of tran scriptional permissiveness (Cha-Molstad et al., 2004). It was previously determined that the HSV1 LAT promoter contains a functional cAMP response element (CRE) with complete homology to the proenkephalin CRE (Leib et al., 1991). This element, referred to herein as the -43CRE due to its starting location relative to the LAT transcriptional start site, was shown to be inducible upon appl ication of known modulators of intracellular cAMP levels, and it was also shown to bind the wild-type form of the CRE binding (CREB) protein in vitro (Leib et al., 1991; Millh ouse et al., 1998). Interestingly, when mobilityshift assays were performed usi ng PC-12 cell nuclear extracts, resu lts suggested that a protein or proteins other than CREB can bi nd specifically to the LAT -43CRE (Leib et al., 1991); however, this observation was not further explored. The role of the -43CRE in reactivation was also examined in vivo using a recombinant virus, in which the mutated binding site was predicted to provide le ss than 10% CREB binding (Bloom et al., 1997). When the recombinant virus was tested in the rabbit eye model, epinephrine induction yielded reactivation that wa s intermediate (58%) between wild-type strain 17 syn + (78%) and promoter-deletion mutant 17 Pst (19%) (Bloom et al 1997). Spontaneous reactivation levels were similar betw een the -43CRE mutant virus and 17 Pst (19% and 16%, respectively) (Bloom et al., 1997). In the rabbit st udies, as well as in studies performed using the mouse hyperthermic stress model, the mutation in the -43CRE mutant virus did not appear to affect establishment or maintenance of latency (B loom et al., 1997; Marquart et al., 2001). Thus, the -43CRE seems to have a role in react ivation. Because epinephrine can stimulate adrenergic receptors to begin the cAMP cascade, it seems plau sible that the induction of reactivation is tied to th e responsiveness of the -43CRE. In fact, when the -adrenergic receptor-

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47 blocker, propanolol, was injected into mice la tently infected with HSV-1, the appearance of infective virus was decreased significantly in the tear film, cornea, and trigeminal ganglia (Gebhardt and Kaufman, 1995), and when it was used to treat laten tly-infected rabbits, spontaneous reactivation was reduced (Kaufman et al., 1996). Therefor e, the cAMP response pathway may play a role in HSV-1 reactivation from latency. Approximately 40 base pairs upstream of the -43CRE exists a second CRE, which will be referred to as the -83CRE. This elem ent was identified through chloramphenicol acetyltransferase (CAT) activity a ssays of various promoter dele tion mutants and was suggested to convey cell-specificity, as determined through testing in C1300 mouse neuroblastoma cells and L929 mouse fibroblast cells (Kenny et al., 1994). Specifically, when promoter sequences including the -83CRE were adde d to the neuronal cells in pl asmid-based transient expression assays, a threeto fourfold in crease in promoter activity was ob served, while no effect was seen in nonneuronal cells (Kenny et al., 1994). U pon further examination of the -83CRE, it was determined through electrophoretic mobility sh ift assays (EMSA) using C1300 cell nuclear extracts that although the site can bind both CREB-1 and CREB -2, it binds CREB-2 with much higher affinity (Millhouse et al., 1998). CREB-2, when over-expressed in vitro causes a significant repression of CRE-mediated transcri ption due to a lack of phosphorylation sites (Karpinski et al., 1992). Thus, in the HSV-1 LAT promoter, it is possible that the site plays a role in the repression of LAT transcription that is observed during early times post-explant (Amelio et al., 2006). One might envision the LAT promoter as a mol ecular switch that controls LAT expression to regulate the transition between latency and reactivation, especia lly since transcription of ICP0, an IE gene, occurs downstream of the LAT promoter and in an antisense orientation to the LAT,

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48 with the potential for overlap of both transcripts. The ICP0 protein has several defined functions, including transact ivation of various cellular and vira l genes, host protein degradation, and viral localization to ND10 cellu lar structures (reviewed in Ever ett, 2000). ICP0 mutants are viable and can establish latent infection, although they display re duced replication in cell culture at low m.o.i. (Stow and Stow, 1986; Sacks and Sc haffer, 1987; Everett, 1989) and inefficient reactivation from latency (Leib et al., 1989; Cai et al., 1993). In terestingly, the inefficient growth by the ICP0 mutants can be restored to near wild-type le vels when grown at high m.o.i. in cell culture (Sacks and Schaffer, 1987). The w ild-type ICP0 promoter can be activated in response to stress, as demonstr ated through stress-stimulating experiments in ICP0 reporter transgenic mice (Loiacono et al., 2003). A dditionally, the chromatin surrounding the ICP0 promoter becomes more transcri ptionally permissive within f our hours of murine DRG explantinduced reactivation (Amelio et al., 2006). While it appears that there may be some role for ICP0 in reactivation, it is not clear whether ICP0 plays a cri tical role in facilitating IE transcription at very early tim es during reactivation, or whethe r it functions as a general transactivator of transc ription that enhances th e reactivation process. Because of the potential importance of the -83CRE in regulating the LAT promoter and possibly in facilitati ng reactivation the experiments de scribed here were designed to characterize and define the func tion of the HSV-1 LAT promoter -83CRE site. Creation of a recombinant virus with an eight base-pair muta tion in the -83CRE site of the LAT promoter allowed the relevance of the site in the acute, la tent, and reactivation phase s of the viral lifecycle to be examined.

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49 Materials and Methods Plasmid Generation, Mutage nesis, and Purification Plasmid pNG1 was generated through the subc loning of the 1.2 kiloba se fragment of the LAT promoter through the 5’ exon contained between the Dra I and BstE II restriction enzyme sites (nt 118,002–119,202) into pBluescript II. The resulting plasmid was subjected to site -directed mutagenesis of the -83CRE site (AATTACA) to a Bgl II restriction enzyme site using Stra tagene’s Quikchange II Site-Directed Mutagenesis kit. Nucleotides were mutated in gro ups of four using the foll owing sets of primers: P1Sense—GCA GAC GAG GAA AAT AAA ACA GAA TCA CCT ACC CAC GTG GTG CTG TGG; P1Antisense—CCA CAG CAC CAC GTG GGT AGG TGA TTC TGT TTT ATT TTCCTC GTC TGC; P2Sense—GCA GAC GAG GAA AAT AAA ACA GAT CTT CCT ACC CAC GTG GTG CTG TGG; P2Antisense—CCA CAG CAC CAC GTG GGT AGG AAG ATC TGT TTT ATT TTC CTC GTC TGC. The mutagenesis reaction was performed according to manufacturer’s inst ructions using 50 ng of starting plasmi d, and thermal cycler conditions were as follows: 1 cycle of 30 sec. at 95C and 16 cycl es of 30 sec. at 95C, 1 min. at 55C, and 4 min. at 68C. Because the mutated site created a Bgl II restriction enzyme site not present in the parental DNA, mutated plasmid DNA was subjected to restriction endonuc lease digestion with that enzyme to further verify that the corre ct mutation was obtained. Additionally, plasmids were sequenced for verificat ion of the correct mutation. Upon confirmation of the desired mu tation, the plasmid DNA was grown in E. coli cells and purified using a cesium ch loride gradient (Garger et al., 1983). Briefly, the plasmid was grown in 2x YT media containing ampicillin, pe lleted, and resuspended in glucose buffer (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM Na2EDTA). Bacterial cells were lysed in 20 mg/ml lysozyme and lysis solution (0.2 N NaOH, 3% SDS, and water to volume). Potassium

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50 acetate solution (5 M potassium acetate and 11.5% gl acial acetic acid) was added and incubated in an ice water bath for 15 min. The sample was centrifuged, the supern atant was filtered and chloroform:isoamyl alcohol (24:1) extracted, and the plasmid precipitated with isopropanol. The pellet was resuspended in 1x TE (10 mM Tris-H Cl, pH 8.0, 1 mM EDTA), combined with 4 g cesium chloride, and 4 mg ethidium bromide. After overnight cen trifugation at 44,000 rpm (176,284 x g) in a VTi 65.2 vertical rotor, the plasmid DNA band, visible upon exposure to ultraviolet light, was removed using a syringe a nd extracted against isoamyl alcohol to remove the ethidium bromide. DNA was precipitated, phenol:chloroform extracted, precipitated again and resuspended in 1x TE. Cells and Viruses L7 cells, a Vero cell line containing ICP0 stably transfected (Samaniego et al., 1997), were a gift from the lab of Neal DeLuca. Rabb it skin (RS) and L7 cells were grown at 37C in the presence of 5% CO2 in minimal essential media (MEM) supplemented with 5% calf serum (RS cells) or 10% fetal bovine serum (L7 cells), 292 g/ml L-gl utamine, and antibiotics (250 U/ml penicillin and 250 g/ml streptomycin). Neuro-2A (C1300) cells were obtained from the American Type Culture Collection and gr own at 37C in the presence of 5% CO2 in MEM supplemented with 10% fetal bovine serum, 292 g/ml L-glutamine, antibiotics (250 U/ml penicillin and 250 g/ml streptomycin), and 1 x Non-Essential Amino Acid s (Mediatech, Inc.). For transfections, RS cells were subject to overnight serum-starvation at 31.5C (5% CO2) with MEM supplemented with 1.5% fetal bovine serum, in addition to L-glutamine and antibiotics as described above. HSV-1 strain 17 syn + was obtained as a low passage stock from J. Stevens. 17 PstStuffer (replacement of nucleotides 118,666 to 118,869 of 17 syn + with the Kpn ISac I fragment

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51 of pBluescript’s multiple cloning site [MCS]) wa s constructed by D. Bloom. -83CRE and F8-1 were constructed as described below. DNA Isolation for Transfections 17 Pst-Stuffer virus was used as the backbone for the construction of the -83CRE viral recombinant containing the site-directed mutation of the -83CRE. The -83CRE recombinant was used in the constructi on of the rescuant, F8-1. In orde r to prepare the viral DNA used for transfections, virus was cultivated on RS cells at an m.o.i. of 0.01. Upon appearance of cytopathic effect (CPE), inf ected cells were harvested, centr ifuged, and the pelleted cells resuspended in hypotonic lysis buffer (10 mM Tris, pH 8.0, 10 mM EDTA, 0.5% Nonidet P-40, 0.25% NaDOC). After 5 min. incubation on ice, the sample was centrifuged, the supernatant removed and incubated with 1 mg/ml Pr oteinase K and 1% SDS for 1 h at 50 C; 1 mg/ml Proteinase K was added after the initial in cubation and incubated for 1 h more at 50 C. The sample was extracted with phenol, chloro form, and isoamyl alcohol. Viral DNA was precipitated by the additi on of 0.1 vol 3 M NaOAc, followed by th e very slow addition of 2 vol ice cold 100% ethanol. The DNA wa s spooled, removed, air-dried and resuspended in 1x TE. Virus Construction and Plaque Purification Plasmid DNA was transfected with viral DNA to allow for recombination. The mutated -83CRE plasmid DNA was first digested using BspE I and BsaB I and was then gel purified. Approximately 4 g of the purified, linearize d plasmid DNA was combined with varying amounts of 17 Pst-stuffer DNA (2 g, 4 g, 8 g, and 16 g) in a final volume of 225 l TNE buffer (10 mM Tris, pH 7.4, 1 mM EDTA and 0.1 M NaCl) plus 25 l CaCl2. Rescuant F8-1 was constructed by combining -83CRE viral DNA with plasmid pAatII (nt 4817-9271 of 17 syn +). The DNA was precipitated by the addi tion of 2 x HEPES while blowing bubbles through a pipet and allowed to incu bate for 20 minutes at room temperature. This transfection

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52 mix was then applied to RS cells which had unde rgone an overnight serum-starvation (described above), and this was allowed to incubate at room temperature fo r 30 min. After this incubation, MEM supplemented with 1.5% fetal bovine serum was added to the cells and incubated for 4 hours at 37C in the presence of 5% CO2. This incubation was followed by the addition of hypotonic lysis buffer (10 mM Tris, pH 8.0, 10 mM EDTA, 0.5% Nonidet P-40, 0.25% sodium deoxycholate) directly to the monolayer, followed by three washes with media. Supplemented MEM containing 5% calf serum was added to the cel ls, which were then in cubated at 37C in the presence of 5% CO2 for 3-4 days (when CPE was observed). Screening for recombinant viruses was performed by picking plaques followed by dot blot hybridization. The virus resulting from the transfection was diluted to yield well-separated plaques (10-4 through 10-7), and 0.5 ml of these dilutions pl ated onto confluent monolayers of cells in 60-mm tissue culture dishes (two to three dishes per diluti on). After 1 hour of adsorption, monolayers were overlayed with 0.75% agarose in 1x supplemented media, and the dishes were then incubated until plaques were visible (2-3 days). To assist in visualization of plaques, dishes were counterst ained with a 1:30 dilution of 3.3 g/L Neutral Red (Sigma) in unsupplemented media for approximately 4-6 hour s. The liquid overlay was removed, and plaques were picked using sterile Pasteur pipettes. After gently aspirating into the pipette, the plaques were expelled into a 96well dish containing 150 l media per well. Once plaques were picked, the dish was frozen at 80C and then thawed in the tissue culture incubator. 50 l of the plaque suspensions were inoculat ed onto confluent cells in a 96-we ll dish and allowed to adsorb for 1 hour. At the end of the incubation, supplemented media was added to each well, and the dishes were incubated until 100% CPE was observe d (approximately 3 days). A Millipore dotblot apparatus was used to transfer 50 l of infected cells onto a nylon membrane, which was

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53 washed with 200 l of a solution containing 1.5 M NaCl and 0.1 M NaOH, followed by 200 l of a solution containing 0.2 M Tris, pH 7.5, and 200 l of a solution containing NaCl, NaH2PO4, and EDTA. Once the solutions were through the apparatus, the membrane was baked for one hour at 80C. Dot blots were probed with radioactively labeled random-primed DNA probes prepared using the Pst-Pst fragment of the HSV-1 LAT promoter, th e MCS fragment of pBluescript, or a 22-mer encompassing the wild-type -83CRE of th e LAT promoter. Probes were labeled with [ -32P]dCTP using the Rediprime II Random Prime Labelling System (Amersham Biosciences), according to the manufacturer’s protocol. To screen for the recombinant -83CRE viru s, the membrane was prehybridized for 4–5 hours at 62.5C with a solution containing 3 M Na Cl, 0.3 M sodium citrate, 50 mg/ml nonfat dry milk and 2 l/ml Antifoam A, the labeled probe added to the prehybridiz ation solution and the dot blot hybridized overnight. The membrane wa s then washed at room temperature once for 5 min. with a wash solution containing 0.3 M NaCl, 0.06 M Tris-HCl, and 0.002 M EDTA and twice for 5 min. each with a wash soluti on containing 0.03 M NaCl, 0.006 M Tris-HCl, and 0.0002 M EDTA. The blot was dried and e xposed to X-ray film overnight. To screen for the rescuant virus, F8-1 tetramethyl ammonium chloride (TMAC) hybridization was used. The membrane was prehybridized for 1 hour at 58C with a hybridization solution consisting of 3 M TMAC, 0.1 M NaPO4, pH 6.8, 1 mM EDTA, pH 8.0, 5 x Denhardt's Solution, 0.6% SDS, and 100 g/ml denatured Salmon Sperm DNA. For the hybridization, the hybridization solution was replaced with fresh solution, and a labeled 22-mer probe identical to the wild-typ e promoter region encompassing the -83CRE site was hybridized to the membrane for 24 to 48 hours at 58C. The membrane was washed twice at room

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54 temperature with Wash #1 (3 M TMAC, 50 mM Tris, pH 8.0, 0.2% SDS), once with Wash #1 at 60C for one hour, and twice at room temperat ure with Wash #2 (2 x SSPE, 0.1% SDS, 1 mM EDTA, pH 8.0). The membrane was then dried and exposed to X-ray film. F8-1 was one of two independent plaques identified from the initial screen and purified through 4 rounds of plaque purification. PCR Analysis The LAT promoter regions of the recombin ants were analyzed by PCR performed on viral DNA that was isolated as described above (see Cells and Virus Cultivation/DNA Isolation for Transfections). Amplifica tion reactions contained 1x GoTaq Green Master Mix (Promega), 600 ng each of primer Upfragment, 5’ —CGA GGA ACA ACC G AG GGG AAC (nt 118,305118,325) and Downfragment, 5’—CTG AG A TGA ACA CTC GGG GTT ACC (nt 119,179119,202), 50 ng of viral DNA and nuclease-free water to a final volume of 50 l. The DNA was amplified using an Ericomp thermal cycler (San Diego, CA) with 2 min. at 95C (one cycle), 3 min. each at 94C, 55C, and 72C (one cycle), and 1 min. each at 94C, 55C, and 72C (three cycles). PCR products were visu alized on 1% agarose gel containing ethidium bromide. Growth Curves In order to assess replication of the -83CRE recombinant in various cell lines, the virus was inoculated at a multiplicity of infection (m.o.i.) of 0.001, 0.01, or 5. The virus, along with its rescuant (F8-1) and the wild-type 17 syn + virus, was used to infect confluent RS cells, L7 cells, or Neuro-2A cells grown in 35-mm cell culture dishes. Ti me (hours post-infection) was monitored starting one hour after the inoculum was added to the cells. Infected cells were harvested by gentle scraping of the dishes at 0, 8, 24, 48, 72, and 96 h.p.i. for multi-step growth curves (low m.o.i.) or 0, 8, 24, and 48 h.p.i. for si ngle-step growth curves (high m.o.i.). After one freeze-thaw, viral titers were determined.

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55 Mouse Survival Assay Six to 8 week old female ND4 Swiss mice (H arlan Sprague Dawley, Inc.) were infected via the footpad with 500 pfu, 5,000 pfu, or 50,000 pfu. Briefly, mice were injected with saline in the rear footpads to soften th e keratinized epithelium. Three to four hours later, mice were anesthetized intramuscularly wi th 0.01 to 0.02 ml of a cocktail of ketamine (30 to 45 mg/kg), xylazine (7.5 to 11.5 mg/kg) and acepromazine (2.5 to 3.75 mg/kg). During this time, the footpads were abraded with an emery board an d virus was applied. Vi ral absorption occurred during the time that the mice remained under an esthesia (approximately 45–60 minutes). The number of mice surviving throughout the ac ute phase of infection was assessed. Intracranial Inoculation as an Assay for Neurovirulence In order to assess the neur ovirulence of the recombinant virus, ND4 Swiss mice were anesthetized using isof luorane and inoculated intracranially with 10 l of dilutions of virus using a 27 gauge needle. Doses given were 10 pfu and 100 pfu. Mice were monitored for survival over the acute phase of infection. DNA Extraction and Analysis of Course of Infection Mice were infected via the footpads w ith 5,000 pfu/mouse (8 hour sample) or 10,000 pfu/mouse (2 and 4 day samples) of either 17 syn + or -83CRE virus. At 8 hours, 2 days, or 4 days post-infection, 4-5 mice from each group were sacrificed, and their feet, DRG, and spinal cords were snap-frozen in liquid nitrogen. Tissu e samples were homogenized in 4 ml (feet) or 0.2 ml (DRG and spinal cord) TES (10 mM Tr is, pH 7.4, 0.1 M NaCl, 1 mM EDTA) with Duall glass dounces and treated with 1% SDS and 1 mg/m l Proteinase K overnight at 50C. Samples were phenol:chloroform:isoamyl alcohol extract ed, chloroform:isoamyl alcohol extracted, and ethanol precipitated. After air-drying, the DNA pellet was resuspended in 1xTE. Spinal cord and DRG samples were diluted 1: 10 and foot samples were dilute d 1:100 for use in real-time

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56 PCR. HSV-1 genome equivalents were determined through calculation of the relative quantity of PCR amplification of the HSV-1 polymeras e gene normalized to the PCR amplification products of the cellular gene target, XIST. RNA Isolation and Reverse Transcription for Acute RNA Levels in Cell Culture RS or Neuro-2A cells were seeded for next day confluency in 35-mm tissue culture dishes. Upon confluency cells were inf ected at an m.o.i. of 0.01 with either 17 syn + or -83CRE virus. One ml of viral inoculum in MEM was a pplied to each dish, allowe d to adsorb into the cells for one hour, and then replaced with fresh s upplemented MEM. After this point, cells were harvested for RNA at 2, 4, 6, or 8 hours post-infec tion (with timing started after the application of fresh media) using 1 ml Trizol, which was tritur ated to lyse cells. Samples were transferred to Eppendorf tubes, incubated at RT for 5 min., a nd 0.2 ml chloroform was added to each tube, vortexed and incubated at RT for 2 min. Tube s were centrifuged at 12,000xg for 15 min. at 4C before the aqueous phase was transferred to a new tube and the RNA was precipitated by the addition of 0.5 ml isopropanol. Samples were in cubated at RT for 10 min. and centrifuged at 12,000xg for 10 min. at 4C. After the removal of the supernatant, the RNA pellet was washed with 1 ml 70% EtOH and spun at 7,500xg for 5 min. at 4C. This supernatant was removed and the pellet was air-dried briefly before be ing resuspended in 45 l nuclease-free H2O. Upon resuspension of the precip itated RNA, DNase treatment was performed using Turbo DNA-free (Ambion) according to the manufacturer’s instruction. Reverse transcriptions were performed with Omniscript reverse transcriptase (Qiagen) in reacti on volumes of 20 ul. Briefly, reactions contained Omniscript 1x buffer, 0.5 mM each dNTP, 1 M random decamer primer (Ambion), 10 units/ul SUPERase-In (Ambion), 1 g RNA, 8 units Omniscript reverse transcriptase, and RNase-free water to volume. Additionally, RNA contro ls (“No RT” controls)

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57 were performed using the same concentration of RNA in water. Reactions were incubated at 37C for one hour. Taqman Real-time PCR Analysis cDNA or DNA was amplified by real-time PC R using TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems) and FAM-labeled TaqMan target-specific primers/probes (Applied Biosyste ms, Inc.) (Table A-1). Reactio ns were run in triplicate in concentrations recommended by th e manufacturer. Primer and pr obe sequences are listed in Appendix A. PCR was performed and analyzed using Applied Biosys tems 7900HT Sequence Detection Systems. Cycle conditions used were as follows: 50C for 2 min. (1 cycle), 95C for 10 min. (1 cycle), 95C for 15 sec., and 60C for 1 min. (40 cycles). Threshold values used for PCR analysis were set within the linear range of PCR target amplification. Average cycle threshold (Ct) values determined in triplicate were averaged, and th e relative quantity was calculated using a standard curve specific for the pr imer/probe set of interest. Briefly, Ct values for 10-fold dilutions of DNA (viral or cellular) of known concentration were determined and graphed as a function of dilution. The equation of the resulting lin e was used to extrapolate the relative quantity of the sample of unknown concentration. RNA Isolation and Reverse Transc ription for Explant Studies Mice (infected as described above with 400–500 pfu of virus) were euthanized by an overdose of isoflourane, followed by cervical di slocation. Dorsal root ganglia (DRG) from groups of two mice (8 ganglia per mouse) were re moved as quickly as possible (3 to 5 minutes per mouse) and placed in 0.5 ml RNA Later (Ambi on). RNA was isolated from the tissue using the method of Chirgwin, et al (1979). Briefly, DRG were homogenized in guanidine thiocyanate solution and 100 l removed for DNA isolation. The homogenate was layered on a 5.7 M cesium chloride cushion and centrifuged overnight at 30,000 rpm (111,132 x g) in a SW

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58 41 Ti rotor. The supernatant was aspirated fr om the RNA pellet, which was briefly dried and then resuspended in nuclease-free water. R NA was precipitated overnight using 0.1 vol 3 M sodium acetate and 2 vol 100% ethanol. The DNA fraction of the sample was isolated as described by Kramer and Coen (1995). Briefl y, the DNA was precipitated overnight with 0.1 vol 3 M sodium acetate and 2 vol 100% ethanol. The resulting pellet was resuspended in 50 l of DNA resuspension solution containing: 0.2 g/m l proteinase K, 0.02% Tween 20, 1 x PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton X-100), and 1.5 mM MgCl2. The DNA was incubated at 65C for 2 h, 80C for 20 min., and 94C for 10 min. (to inactivate enzyme). Because of the pres ence of precipitate, the DNA sample was diluted 1:10 prior to use in real-time PCR analysis. Upon resuspension of the pr ecipitated RNA, DNase treat ment was performed using Turbo DNA-free (Ambion) according to the manufact urer’s instruction. Reverse transcriptions were performed with Omniscript reverse transc riptase (Qiagen) as described above (see RNA Isolation and Reverse Transcription for Ac ute RNA Levels in Cell Culture). ChIP Analysis Dorsal root ganglia were removed and pooled from 3 mice per time point. After incubating in media at 37C for a given amount of time (0, 0.5, 1, 2, or 4 h post-explant), ganglia were homogenized in 0.5 ml phosphate-buffere d saline (PBS) in the presence of protease inhibitors (1 g/ml aprotinin, 1 g/ul leupept in, and 1 mM PMSF). DNA-histone complexes were cross-linked by the addition of 37% formaldehyde to a final concentra tion of 1%. After the addition of 0.128 M glycine, the sample was pelleted and washed three times with PBS containing protease inhibitors as described above. Samples were pelleted, resuspended in SDSlysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl), and sonicated (Fisher Sonic Dismembrator 100) to yield fragments of 500–1000 bp (setting 4, 2 bursts of 40 sec. each

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59 followed by 1 burst of 20 sec.). Sonicated samples were pre-cleared with Salmon Sperm DNA/Protein A Agarose beads (Upstate), and hi stone-DNA complexes were immunoprecipitated overnight with 3.5 g/ml of anti-acetyl-Histone H3 (Upstate). Prior to th e wash steps, 25% of the sample was removed and retained as the “unbound” fraction. Complexes were washed with Low Salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl), High Salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), LiCl (0.25 M LiCl, 1% Nonidet P-40, 1% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl), and TE (10 mM Tris-HCl, 1.2 mM EDTA) wash buffers prior to the immune complexes being eluted from the agarose beads with elution buffer (1% SDS, 0.1 M sodium bicarbonate). DNA (bound sample) was de-crosslinked from histones with 10 l/ml 5 M NaCl and then treated with 20mg/ml RNase A and 40 g/ml Proteinase K. DNA (bound and unbound fractions) wa s purified using a QIAquick PCR Purification kit (Qiagen) before analysis by Taqman real-time PCR. Rabbit Reactivation One to 2 kg New Zealand White rabbits were in fected and housed at the Louisiana State University Health Science Center’s Animal Facility. Each rabbit eye received topical proparacaine-HCl anesthetic prior to corneal scarification. Rabbits were infected with either 17 syn + or -83CRE virus inoculum at 200,000 pfu/eye. At days 3, 5, and 7 post-infection (p.i.), the infections of the rabbit eyes were monitore d by slit lamp examination for the presence of dendrites on the corneas. Clinical scores (1 to 5, with 5 being th e most severe) were assigned to reflect the relative surface area of the cornea covered with dendrites as a means of assessing the severity of the infection. After 28 days p.i., rabbits were anesthetized with ketamine /xylazine and a solution of 0.015% epinephrine in sterile water (dissolved by the addition of one drop of HCl) was iontophoresed into the rabbit co rneas by applying current for 8 minutes at 0.8 mAmps. At

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60 specified times post-iontophoresis rabbits were euthanized w ith sodium pentobarbital and decapitated for removal of TG. The TG were re moved as rapidly as possible (5 to 15 min per rabbit). Results The -83CRE virus construction was not stra ightforward. After a mutation was made to the -83CRE site in a plasmid containing the LA T promoter and verified through sequencing, the mutant plasmid was allowed to homologous ly recombine with DNA from virus 17 Pst-Stuffer, a LAT-negative mutant containing a bacterial stu ffer in place of the core LAT promoter. To ensure that the recombinant used for characte rization experiments was not made up of a mixed population of viral genomes (e.g., the parental st uffer and the nascent recombinant), the virus was plaque-purified, which permits selection for purity. Although the plaque purification appeared to produce a pure recombinant virus af ter approximately seven rounds of purification based on the dot-blot hybridizations, when th e promoter region was amplified through PCR (Figure 3-1), two products appeared for the suppo sed “pure” recombinant (Figure 3-2A). The product of higher molecular weight corresponded to the recombinant, while the lower molecular weight product was representative of the parental virus, 17 Pst-Stuffer. This suggested that the virus was persisting as a mixed popul ation of recombinants and wild -type virus or more likely as a “single-sided” recombinant, in wh ich only one of the two long repeats (RL) contained the LAT promoter -83 CRE mutation (see Figure 3-2). Si nce the lab had never had a recombinant that needed more than 4 rounds of plaque-purificat ion to achieve purity and because the PCR data was consistent with a single-sided virus, this s uggested that there might be a bias against making the -83CRE mutation in both copies of the LAT. Because the -83CRE mutation could potentially disrupt regulation of LAT expression and affect ICP0 in the process, the virus was plaque-purified on L7 cells, a Vero-cell derived cell line, in which ICP0 is stably expressed

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61 (Samaniego et al., 1997). Since the parental 17 Pst-Stuffer virus used in the transfection contained a pBluescript multiple cloning site (M CS) stuffer, the MCS fragment was used as a negative hybridization cont rol. After four rounds of plaque purification on L7 cells, dot blot for the MCS stuffer indicated that th e region did not exist in the -83 CRE virus (not shown), and the PCR product corresponding to the parental vi rus was no longer visibl e, suggesting that complementing ICP0 with L7 cells allowed a viru s with the mutated -83CRE in both copies of the LAT promoter to be purified to homogeneity (Figure 3-2B). As will be discussed further later, it was determined upon characterization of the -83CRE virus that a second site mutation also occurred somewhere in the viral genome. The -83CRE Recombinant’s Replication Is Alte red during the Lytic Phase of the Infection After the -83CRE recombinant virus was purified and the correct mutation in the LAT promoter confirmed through PCR an alysis and sequencing, it was te sted for replication in cell culture. Wild-type 17 syn + and -83CRE virus were used to in fect RS cells, L7 cells, and Neuro2A cells at the low m.o.i. of 0.01 to allow for mul tiple rounds of replication. As shown in Figure 3-3A, the -83CRE displayed approximately 10fold more efficient replication than 17 syn + during the first 8-24 hours of infection when assayed on RS cells, which are epithelial in origin. The opposite was observed for the -83CRE’s growth on Neuro-2A cells, which produced yields of approximately 10-fold less than 17 syn + throughout the times tested (Figure 3-3B). Since the mutant was purified on L7 cells, it was tested for growth on that cell line as well. Providing excess ICP0 had no major effect on the -83CRE mu tant’s replication, sinc e relative yields on L7 cells were the same as on RS cells, with the muta nt again replicating slig htly more efficiently than the wild-type virus early in the infection (Figure 3-3C). Since the recombinant was not able to be pur ified until it was plaqued on L7 cells, an ICP0-complementing cell line, the possibility existed that ICP0 function was altered in the

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62 -83CRE. ICP0-deleted viruses show a multiplicit y-dependent effect on re plication, in which the mutant grows very inefficiently at low m.o.i. but near wild-type leve ls of replication are restored at high m.o.i. (Sacks and Schaffer, 1987). To determine whether the -83CRE mutation had a multiplicity-dependent effect on re plicative yields, single-step growth curves (m.o.i. of 5) were performed using RS and Neuro-2A cells. As shown in Figure 3-4, the -83CRE recombinant shows a similar effect to that observed for low m.o.i. on RS cells but the decrease d replication on Neuro-2A cells that was seen for the low m.o.i. infection was not observed. In fact, the -83CRE demonstrated slightly enhanced replication (a pproximately 5-fold) rela tive to wild-type at 12 hours p.i. when grown on Neuro-2A cells. Thus, these data suggest a multiplicity-dependent effect of the -83CRE recombinant on viral grow th on the Neuro-2A cell line, but not on the RS epithelial cells. The -83CRE Recombinant Displays Impaired Replication and Spread in the Nervous System of the Mouse Because the -83CRE mutant virus showed so me variation from wild-type in replication assays, it was important to test the virus in th e mouse to determine what biological effects the mutation would have. Ten ND4 Swiss mice per dos e per virus were infected with 500, 5000, or 50000 pfu of either 17 syn + or the -83CRE virus and were monitored closely during the acute phase of infection (first three w eeks of infection). Th e survival of mice at the varying doses is shown in Figure 3-5 and in Table 3-1. While 17 syn + resulted in 0–30% survival for the doses tested, mice infected with the -83CRE virus displayed a 90% survival rate for all doses, indicating an LD50 of >5x104 pfu. Because of this dramatic deficit in virulence following infection via the mouse footpad, we sought to de termine whether the defect in virulence was a result of decreased replication on the epithelium of the foot and/ or within the nervous tissue.

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63 To determine whether the -83CRE virus exhib ited a replication defect in nervous tissue, mice were intracranially (i.c.) inoculated with the -83CRE virus to determine its virulence following direct delivery to the CNS. In this assa y direct injection of 10 or 100 pfu of virus into the brain of mice was performed. Mice were cl osely monitored for death and sacrificed once CNS involvement was observed (determined by para lysis, erratic movement, and/or inability to right itself). As shown in Tabl e 3-2, the mutant -83CRE virus e xhibited a slight decrease in neurovirulence following i.c. inoculation when compared to 17 syn +. These results suggest that the -83CRE virus was attenuated in its ability to replicate in the nervous system, but it could kill mice if delivered to the brain directly at higher doses. The relative avirulence seen following footpa d inoculation could have been due to a cumulative effect of slightly less efficient repl ication at each node of the nervous system in which the virus replicates on its path from the foot to the brain. It also was possible, however, that the virus replicates less efficiently in the ep ithelium of the foot of th e mouse and not just in the nervous tissue. In order to differentia te between these possibi lities a tracer study was performed. Mice were infected via the footpad rout e and sacrificed at 8 hours, 2 days, or 4 days p.i. Foot, spinal cord and DRG were assessed for relative numbers of viral genomes. As shown in Figure 3-6A & B, replication in the foot is equivalent for all times examined. However, once the -83CRE mutant virus reaches the DRG, rep lication is reduced by approximately 2.5to 5.5fold relative to 17 syn +, while replication in the spinal cord is reduced by approximately 5to 8fold (Figure 3-6C, D). These re sults support the viral growth curve data, as well as the mouse infection data, suggesting that the -83CRE mutant exhibits a reduced ability to replicate within neurons.

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64 The -83CRE Virus Contains a Second Site Mutation Since the virulence phenotype observed for the -83CRE mutant was quite dramatic, it was necessary to confirm that the -83CRE recombinan t virus’s phenotype was indeed due to the 8-bp mutation in the LAT promoter. To do this the mutation was rescued by th e transfection of wildtype LAT promoter DNA with the -83CRE virus. If the -83CRE site was the only mutation in the virus, restoration of the w ild-type LAT promoter should re store virulence to the rescued virus. Mice were inoculated via the footpad with varying dos es (500, 5000, 50000 pfu/mouse) of the wild-type, the -83CRE recombinant, or the re scue virus, F8-1. Lit tle to no mortality was observed for the F8-1 rescuant virus at any of the doses tested (Fi gure 3-7). The similar virulence phenotypes of the F8-1 virus and the -83CRE virus suggested a second mutation somewhere in the mutant virus’s genome. Inte restingly, when the F8-1 virus was assayed for replication using a multi-step growth curve on RS cells, there appeared to be a delay in replication efficiency, with decreased replication at 8 hours p.i. but leve ls similar to that of the -83CRE and wild-type at the later times tested (Figure 3-8). This s uggests that rescuing the -83CRE mutation may have altered the ability of the virus to replicate on RS cells, but did not rescue a second mutation that s eems to primarily affect neuron al replication and virulence in vivo Ongoing experiments using subfragments of the viral genome to rescue the virulence phenotype will map the site of the second mutation. Mutation of the -83CRE Resu lts in Wild-type HSV RNA Levels in RS Cells during Acute Infection To further analyze the -83CRE mutant, RS cel ls were infected at an m.o.i. of 0.01 with either 17 syn + or -83CRE. Cells were harvested at acute times for RNA analysis. Random decamer-primed reverse transcription reactions were followed by Taqman real-time PCR to determine the relative quantities of select RNA transcript levels. Relative quantities of viral

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65 targets were normalized to relative quantities of the cellular gene, GAP DH, to account for any variations in cell growth or inf ectivity. As shown in Figure 3-9, there is no significant difference in abundance of transcripts from any of th e gene classes (immedi ate early, ICP4; early, Polymerase; early-late, LAT; true late, glycoprote in C). Furthermore, inhibition of transcription by treatment with cycloheximide or blockade of replication using phosphonoacetic acid showed no difference in transcript accumulation betwee n the wild-type and -83CRE viruses. The -83CRE Virus Expresses LAT during Latency Mice were infected with 400-500 pfu for latenc y. RNA was isolated from the dissected DRG using guanidine thiocyanate isolation and was reverse transc ribed using random decamers. Relative quantities for the LAT 5’ exon and ICP4 obtained through Taqman real-time PCR were normalized to the relative quantity for the cellu lar RNA, XIST. As shown in Figure 3-10, the mutant -83CRE virus expresses the LAT at a de tectable level during latency. Additionally, the relative values (HSV-1 polymerase normalized to cellular control XIST) determined for the back-extracted viral DNA are 0.0183 + 0.0084 and 0.0228 + 0.0107 for 17 syn + and -83CRE, respectively, indicating that levels of establishment of the -83CRE virus are similar to those seen for wild-type 17 syn +. These data indicate that mutation of the -83CRE site does not abolish LAT expression during latency. The -83CRE Virus LAT Promoter Region’s H3 K9, K14 Acetylation Le vels Are Similar to Those of ICP0 during Latency In order to examine the chromatin pr ofile of the -83CRE virus versus 17 syn +, mice infected with 400 pfu of virus were sacrificed and their DRG removed and processed for the ChIP assay. Because CREs are able to recrui t CREB binding protein (CBP), which is a known histone acetyltransferase (HAT) (Ogryzko et al ., 1996), anti-acetyl H3 K9, K14 was used to determine whether mutation of the HSV-1 CRE affects the acetylati on levels of the LAT

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66 promoter and/or the LAT enhancer. It was pr eviously shown that the region encompassing the LAT promoter and 5’ exon is enriched in H3 K 9, K14 acetylation relative to lytic genes (Kubat et al., 2004). As shown in Figure 3-11, that is not observed for th e -83CRE virus. In fact, the LAT promoter region is approximately as hypoacety lated as the lytic gene, ICP0. This suggests an abrogation of the ability of CBP, or so me other factor aff ecting transcriptional permissiveness, to bind the LAT promoter when the -83CRE site is mutated. The -83CRE Virus Reactivates from Latency in the Rabbit with Similar Efficiency as Wildtype 17 syn + Rabbits were infected via the ocular rout e at a dose of 200,000 pf u/eye of wild-type 17 syn + or -83CRE. Slit lamp examination of the ra bbit eyes at three, five, and seven days postinfection revealed no significan t difference in pathology between the viruses (Table 3-3). Furthermore, both viruses had similar mortal ity, with 12/26 rabbits (46%) surviving the 17 syn + infection and 10/20 rabbits (50 %) surviving infection with th e -83CRE virus. To assess reactivation efficiency, once the vi ral infections became latent, the rabbit eyes were subjected to epinephrine iontophoresis and swabbed for eight days post-induction. There was no significant difference in reactivation, with the -83CRE reco mbinant reactivating at near wild-type levels (P=0.4, Table 3-4). Discussion Mutation of the -83CRE Is Unfavorable for Recombination The construction strategy of the -83CRE reco mbinant should have essentially rescued the LAT promoter deletion of the parental 17 Pst-Stuffer by replacing the bacterial stuffer fragment with a viral DNA fragment ( Pst-Pst fragment) containing the LAT tr anscriptional start site, as well as a mutated -83CRE site. Detection of the bacterial stuffer fragment in the parental virus would suggest that the recombination did not oc cur. After eight rounds of purification, the

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67 recombinant was fully positive for the Pst-Pst fragment but also fully positive for the bacterial stuffer fragment. This suggested that both regions were present in the recombinant virus, and this hypothesis was confirmed when the re gion was amplified by PCR (Figure 3-2). Recombination, therefore, appeared to occur in on ly one of the two copies of LAT existing in the viral genome, suggesting that the wi ld-type -83CRE site is important in virus viability. In other words, a -83CRE recombinant with the mutati on in both copies of LAT might prevent normal replication from occurring. The LAT is anti-sense to ICP0 and the 3’ ends of the two transcripts overlap. Because of this, one might hypothesi ze that the -83CRE site normally acts as a transcriptional repressor to control LAT expression at inappropriate times, i.e., times when ICP0 is expressed. When the site is mutated, th e virus can no longer repl icate because of LAT’s interference with ICP0. To address this possibili ty, the virus was plaque purified on L7 cells. L7 cells are a Vero-based cell line that was stably transfected to express ICP0 (Samaneigo et al., 1997). Interestingly, after only three to four roun ds of plaque purification, all plaques were positive for the Pst-Pst fragment while none were positive fo r the bacterial stuffer. Plaque purification of the -83CRE virus may have been successful on L7 cells and not on RS cells for the reason mentioned above. Sin ce the need for ICP0 complementation was only seen during the plaque purification stage, and not in later experiments, LAT regulation is probably important during recombin ation. It may be possible that the -83CRE site acts as a repressor of LAT, acting to prevent LAT transcrip tion from occurring at the same time as ICP0. If so, when the -83CRE site is mutated, expression of ICP0 in trans by the L7 cells is necessary to overcome the effects of LAT misregulation. Alternatively, a need for ICP0 in recombin ation may be caused by the still unidentified second site mutation. If the second mutation affect s a region of the virus that is involved in

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68 recombination and ICP0 must be provided in trans to overcome the effect, it may suggest that ICP0 and the second site inter act during recombination or more likely, that the second site mutation is in ICP0 and that ICP0 itse lf is important in recombination. The -83CRE Recombinant Contains a Second Site Mutation that Contributes to the Avirulence Phenotype When the -83CRE mutant virus was rescue d using the wild-typ e LAT promoter, the avirulence phenotype was not rescued. The mortalit y rate of the rescuant-infected mice was very low, indicating the presence of another mutation somewhere in the -83CRE mutant’s genome. This other mutation, therefore, is likely a major contributor to vi rulence of HSV-1. The second site mutation in th e -83CRE recombinant may ha ve arisen during the viral construction phase. If the mutation in the 83CRE site was unfavorable to viable virus production, a second compensatory mutation may ha ve occurred, which ultimately allowed the recombinant to be made. An example of a sim ilar situation exists wi th neurovirulence gene 34.5 viral recombinants. In eukaryo tic cells part of the host defens e against viral infection is the activation of protein kinase R (PKR) in res ponse to double-stranded RNA. PKR induction in a cell causes eukaryotic translat ion initiation factor 2 (eIF-2 ) to be phosphorylated, which ultimately shuts down protein synthesis. In wild-type HSV-1, the 34.5 gene product can dephosphorylate eIF-2 to prevent cellular shutdown of prot ein synthesis (He et al., 1997). As expected 34.5 mutants are unable to prevent cellular inhibition of protein synthesis (Chou and Roizman, 1992). Rescue of the 34.5 mutation led to the isolation of mutants with a second site mutation in the Us11 gene that causes restoration of the PKR suppressor phenotype observed in wild-type HSV-1 (Mohr and Gluzman, 1996). Us11 interacts with PKR if present before PKR induction to block eIF-2 phosphorylation and therefore, allows protein synthesis to proceed and

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69 the virus to function (Cassady et al., 1998). Th e second site mutation in the -83CRE mutant could be functioning in a similar manner, comp ensating for the -83CRE mutation. While the second site mutation in the -83CRE virus remains unmapped, one can speculate as to its location. Potentially, the mutation coul d lie somewhere in ICP0, affecting its normal function. In ICP0-negative mutants, growth in cell culture is ineffi cient at low m.o.i., but replication is restored to near wild-type levels at high m.o.i. (Sacks and Schaffer, 1987; Chen and Silverstein, 1992). While the -83CRE recombinan t did not show the same effect as ICP0 mutants on RS cells, the results from the grow th curve assays on Neuro-2A cells suggest multiplicity-dependent growth in neuronal cells. Recall that the -83CRE virus replicated less efficiently than wild-type on Neur o-2A cells at a low m.o.i., but th e mutant replicated as well or slightly better than wild-type at the high m .o.i. when grown on Neuro-2A cells. This may indicate that the -83CRE recombin ant is functionally deficient in ICP0 in neuronal cells, possibly due to a lack of necessary interplay between effects of the -83CRE mutation, ICP0, and some neuronal-specific factor. When ICP0 is present at high levels, growth in Neuro-2A cells can occur normally. The -83CRE recombinant appeared to grow more efficiently than wild-type during early infection of RS ce lls at a low m.o.i., suggesting that the -83CRE mutation may compensate for a mutated form of ICP0. The animal experiments performed, in which various tissues were assessed for relativ e viral DNA levels, support this hypothesis (Figure 3-6). When the -83CRE initially infected the mouse foot pad, DNA levels were similar between wild-type and the mutant, but levels of the mutant viral DNA were decreased relativ e to wild-type in the DRG and spinal cord. This could be due to a lo wer effective dose of the virus in those tissues; perhaps the -83CRE mutation is no longer an effective means of compensation for whatever defect is present in ICP0. In other words, when the mutant virus is applied to the footpad at a

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70 high dose, it would not appear to act like an ICP0 mutant, in whic h replication is inhibited at a lower m.o.i. Once the -83CRE virus enters th e neuronal tissues, however the amount of viral production begins to drop. This may be caused by an inability of the mutant virus to utilize some neuronal factor properly and has the end result of decreasing th e effective viral dose to the neuronal tissues to one that is not compatible with efficient growth by an ICP0-defective virus. In the mouse survival experiments, the 83CRE virus was severely attenuated for virulence, corresponding to the finding that ICP0 deletion mutants display decreased pathogenicity in some animal models (Gordon et al., 1990). What is puz zling, however, is the fact that the -83CRE virus established latency an d reactivated from latently-infected rabbits with similar efficiency as wild-type 17 syn +, unlike characterized ICP0 mutants (Wilcox et al., 1997; Halford and Schaffer, 2001). When the chromatin profile was determined for the latent -83CRE in mouse DRG, the LAT promoter and the ICP0 promoter had similar levels of H3 K9, K14 acetylation, which is a marker of transcriptional permissiveness, and both of these regions were less acetylated than the LAT 5’exon. Perhaps the chromatin profile of the -83CRE mutant permitted establishment of latency and reactivati on. In other words, even though ICP0 was still hypoacetylated relative to the 5’ exon, it may have st ill been slightly permi ssive for transcription (which would not be apparent from the qualitati ve results of the ChIP assay), and this would have allowed reactivation to occur. Marker rescue experiments will aid in determining the site of the second mutation. Cell-Specific Factors May Interact with the 83CRE Site to Convey Neuronal Tropism to HSV-1 The results from the viral growth curves and the mouse tracer study indicate differential growth efficiencies of the -83CRE recombinan t on epithelial cells than on neuronal cells. Specifically, the mutant replicates more efficien tly than the wild-type virus on epithelial cells,

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71 while it does not grow as well as wild-type on neuronal cells. This may also explain why the -83CRE is attenuated when mice are infected by the footpad infection route. It seems likely that, although the mutant replicates as efficiently as wild-type at the footpad, the efficiency of replication slows once the mutant reaches the sp inal cord and becomes even less efficient once reaching the DRG. By the time it reaches the brai n, which is the site of encephalitic infection that leads to mortality in mice, there is lit tle infectious virus left to cause damage. When the -83CRE virus was used to infect ra bbits, the virus did not show the virulence deficit that was observed for mice. The rabbits displayed similar levels of corneal pathology as wild-type, suggesting robus t replication in th e corneal epithelia by the -83CRE. Because the site of infection (cornea) in th e rabbit is close to the site of latenc y (TG), the virus has less distance to travel and therefore, the -83CRE virus might also replicate less effectively in neurons in the rabbit, as was seen for the mouse, but because of the proximity to the brain, may still produce enough infectious virus to cause death. Experiments to determine viral yields in epithelia versus neuronal tissue during acute infectio n in the rabbit are in progress. Mutation of the -83CRE Site Does Not Affect Latency and Reactivation While mutation of the -83CRE site affect ed the acute infection of HSV-1, it had no significant effect on the establishment of latency, nor did it affect the ability of the virus to transcribe LAT. This suggests that the -83 CRE site, in conjunction with the second site mutation, does not play a role in recruitment of transcription factors to the region during latency. However, when the chromatin profile was dete rmined, the -83CRE mutant virus displayed a profile unlike that seen for w ild-type. During latency the -83 CRE virus’s LAT promoter was less enriched than the 5’ exon/enhancer region and was also less enriched than in wild-type 17 syn +. This hypoacetylation was almost as low as that of the ICP0 promoter, suggesting a

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72 decrease in transcriptional permissiveness in th e -83CRE LAT promoter resulting from a loss of the binding site. However, the LAT was still detect ed by RT-PCR of latently-infected DRG. Because CRE sites are stress-re sponsive elements, it was origin ally hypothesized that the LAT promoter’s CREs would play a role in reac tivation. However, the -43CRE displayed only an intermediate defect in reactivation when tested in rabbits (Bloom et al., 1997), while the -83CRE virus reactivated at the same frequency as wild-type HSV1. It remains possible that the two elements act in concert wi th each other to cont rol reactivation, since the deletion of both elements in 17 Pst results in a virus that does not reactiv ate from latency in the rabbit (Hill et al., 1990). However, since the deletion in 17 Pst is 202 bp, many important factors besides the CREs are deleted, which is likely the cause of the reactivation-negative phenotype. In order to address possible interactions between the tw o LAT promoter CREs without considering the influence of the other binding sites in the pr omoter, a double CRE mutant would need to be created. While not detailed in this dissertation, both the -43CRE and -83CRE were mutated and transfected with HSV-1 DNA in order to cr eate a recombinant with both CREs mutated (CREDBL). Like the -83CRE, the CREDBL re mained single-sided throughout the plaque purifications on RS cells. Unlike the -83CRE vi rus, the CREDBL was unable to be purified on L7 cells. Only about 50% of recombinants were positive, although single-sided, and none became pure after several more rounds of purifi cation. This, coupled with the -83CRE viral construction difficulties, implies a need for the wild -type -83CRE in at least the acute, replicative stage of the viral lifecycle, during which recombination woul d occur. Since the -43CRE recombinant was constructed by Bloom et al. (1997) with little difficulty, this element is likely not as critical as the -83CRE for lytic viral growth.

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73 The Wild-Type -83CRE May Control LAT during the Acute Infection While the -43CRE showed a slight effect on reactivation, there was no significant effect on reactivation that resulted from mutation of the -83CRE. Instead, the major observations— decreased neuronal replication and attenuated vi rulence in the mouse—are linked to the acute HSV-1 infection. Since it was previously report ed that the -83CRE binds the repressive CREB-2 (Millhouse et al., 1998), the site’s main function may be to control LAT. When HSV-1 enters a cell, the cAMP pathway may be induced, since th e entry of a virus into a cell is undoubtedly a stressful event. Various CREB family member s could be produced, including CREB-2. When this is present at the LAT promoter’s -83CRE, the promoter would be repressed. However, in the event that the site is mutated and CREB2 binding is abrogated, perhaps LAT is no longer controlled and would interfere with ICP0 to affect the acute stag e of infection. For this reason, the -83CRE recombinant was not able to be constructed with the mutation in both repeats of the LAT. Instead, a second site compensatory mutati on was necessary that allowed for the viability of the -83CRE virus. This mutation may have occurred in ICP0, possibly affecting some function that normally controls ICP0 expression. If LAT interference with ICP0 by the -83CRE mutation needed to be overcome, the likely solu tion for the virus might be to upregulate ICP0, providing the -83CRE recombinant with a similar environment as that encountered when grown on L7 cells. By characterizing the second site mutation and determining whether it really does occur in ICP0, the importance of the LAT promoter -83CRE site and how it might interplay with the region of the second mutation can be uncovered.

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74 Table 3-1. Survival of mice (number remaining/ number infected) infected via the footpad with either 17 syn + or -83CRE. Dose (pfu) 17 syn + survival -83CRE survival 500 3/10 9/10 5,000 1/10 9/10 50,000 0/10 9/10 Table 3-2. Survival of mice (number remaining/ number infected) inoculated intracranially with either 17 syn + or -83CRE. Dose (pfu) 17 syn + survival -83CRE survival 10 1/5 4/5 100 0/5 0/5 Table 3-3. Slit lamp examination (SLE) scores of rabbit corneas at 3, 5, or 7 days post-infection. Day post-infection 17 syn + average SLE score -83CRE av erage SLE score P-value* 3 3 + 1.1 3 + 1.2 0.82 5 2.8 + 1.1 2.7 + 1.0 0.5 7 2.3 + 1.1 2.3 + 1.1 0.98 P-value calculated using the Mann-Whitney Rank Sum test. Table 3-4. Reactivation of ra bbits post-epinephrine induction. Virus Percent reactivated eyes (Total positive/total eyes) Percent reactivated swabs (Total positive/total swabs) 17 syn + 54.5% (6/11) 26.1% (23/88) -83CRE 78.6% (11/14) 32.1% (36/112)

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75 Figure 3-1. Diagram of PCR primer locations used in verification of -83CRE mutant virus. Recombination fragment for virus cons truction is shown as a gray bar, w ith the -83CRE site shown as a black box. The LAT transcriptional start site is indicated by an arrow. Primers are designated by arrows and U, Upfragment (nt 118,305– 118,325), or D, Downfragment (nt 119,179–119,202). Figure 3-2. Analysis of PCR products amplified fr om dot blot purification. A) The recombinant virus in an early round of purification shows products corresponding to both the parental and wild-type (17 syn +) virus. Diagram of th e -83CRE recombinant genome shows the two copies of the LAT (arrows) and indicates the presence of the stuffer (green) and the mutation (red “X”). B) The recombinant virus PCR product size corresponds to that of the wild-type virus (17 syn +) in purification on L7 cells. Note the presence of a single ba nd. Parental virus, 17 Pst, is also shown. NTC, no template control.

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76 Figure 3-3. Multi-step viral growth curves. 17 syn + (wild-type) or -83CRE (recombinant) were inoculated at an m.o.i. of 0.01 to allow fo r multiple rounds of replication on A) RS cells, B) Neuro-2A cells, and C) L7 cells.

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77 Figure 3-4. Single-step vi ral growth curves. 17 syn + (wild-type) or -83CRE (recombinant) were inoculated at an m.o.i. of 5 to allow for s ynchronous infection of all cells and a single round of replication on A) RS cells and B) Neuro-2A cells.

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78 Figure 3-5. Percent survival over the course of acute infection of mice (10 per group) infected with either 17 syn + or -83CRE at A) 500 pfu, B) 5,000 pfu, or C) 50,000 pfu.

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79 Figure 3-6. Relative viral genomes (Pol, HS V-1 polymerase normalized to cellular gene, Xist ) for 8 hours, 2 days, or 4 days p.i. of mice. Mice were infected via the footpad with either 17 syn + or -83CRE. A) 8 hours p.i. foot samples (n = 4 mice/virus/time), B) foot samples for 2 days p.i. (left panel) a nd 4 days p.i. (right pa nel), C) DRG 2 days p.i. (left panel) and 4 days p.i. (right panel) (n = 5 mice/virus/time), D) Spinal cord 2 days p.i. (left panel) and 4 days p.i. (right panel) (n = 5 mice/virus/time).

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80 Figure 3-7. Percent survival of mice infected with A) 500 pfu, B) 5,000 pfu, or C) 50,000 pfu of 17syn+, -83CRE, or F8-1 rescuant virus.

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81 Figure 3-8. Multi-step growth curv e (m.o.i. 0.001) on RS cells for 17 syn +, -83CRE, and F8-1.

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82 Figure 3-9. Relative RNA transc ript levels (target normalized to cellular control, GAPDH) during acute infection of RS cells. A) Im mediate-early gene, ICP4, B) early gene, HSV-1 polymerase, C) late gene, LAT, and D) late gene, glycopr otein C (gC). Left panel, no treatment. Middle panel, addition of 50 g/ml CHX to cells along with infecting inoculum. Right panel, addition of 400 g/ml PAA to cells with infecting inoculum. Black bars, 17 syn +. Gray bars, -83CRE recombinant.

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83 Figure 3-10. LAT expression duri ng latency in mouse DRG infect ed with the -83CRE mutant virus. RNA was isolated from DRG of late ntly-infected mice a nd reverse transcribed using random decamers. PCR was perf ormed using LAT 5’ exon primers. Figure 3-11. The LAT promoter of the -83CRE mu tant virus is decreased in histone H3 K9, K14 acetylation relative to wild-type. A) 17 syn +, B) -83CRE recombinant. Relative bound, B, to unbound, U, ratios normalized to those for Xist cellular control are shown for the LAT promoter (L. Pro.), th e 5’ exon, the ICP0 promoter, and ICP27.

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84 CHAPTER 4 OVERALL DISCUSSION While the HSV-1 LAT region is known to f acilitate reactivation, the exact mechanism involved is not known. The overall focus of this dissertation, therefore, was to investigate regulation of the LAT, particularly at the level of the LAT promoter. Fi ndings suggest that (1) an element or elements in the core LAT promoter are essential for a latent chromatin profile that is compatible with reactivation, (2) epinephrineinduced reactivation of latently-infected rabbits does not cause the same effects on chromatin as explant-induced reactivat ion of mouse DRG, (3) the -83CRE site of the LAT promoter is dispen sable for establishment of latency, ability to express LAT, and capacity to reac tivate, even though th e latent chromatin profile differs from that of the wild-type virus, a nd (4) a compensatory mutation generated during the construction of the -83CRE recombinant appears to dramatically affect virulence in the mouse and may be important in regulation of the ly tic infection. These data suppor t the developing view that the LAT promoter may be a part of a complex regulat ory switch that modulates gene expression in a tissue-specific manner both during the acute an d latent periods of HSV-1 infection. The LAT and Chromatin During latency transcription from the HS V-1 genome is not repressed through DNA methylation, but instead, histone tail modifications may be a component of gene regulation (Kubat et al., 2004a). In the mouse the LAT region of the latent viral genome shows enrichment in acetylated histone H3 K9, K14, a marker of tr anscriptional permissivene ss, relative to lytic genes (Kubat et al., 2004a; Kubat et al., 2004b). When the LAT promoter is deleted and the virus is unable to transcribe the LAT, the sa me effect is observed, suggesting that the LAT region contains elements that direct the tran scriptionally permissive histone modifications independently of Pol II activity th rough this region. During earl y times post-explant of murine

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85 DRG infected with wild-type virus, there is ch romatin remodeling of the LAT region to a more transcriptionally non-permissive state and decreased LAT leve ls between 2 and 3 hours postexplant (Amelio et al., 2006). A ma jor goal of this dissertation pr oject was to extend the results obtained for the mouse to the rabbit eye model, wh ich more closely mimics clinical reactivation of HSV-1 in humans. In the rabbit, the ability to express at leas t some regions of the LAT correlates with the ability to reactivate from latency. When the ch romatin profile was assessed for a LAT-positive (wild-type) and a LAT-negative (promoter-deletion mutant) virus, two different profiles were observed, unlike what was seen in the mouse. While the wild-type virus displayed a similar latent profile to that of the latently-inf ected mouse, the LAT-negative virus displayed dramatically more enrichment in dimethyl ated H3 K4, a marker of transcriptional permissiveness, in the region just upstream of the deletion than th e 5’exon/enhancer. Levels of dimethylation in the LAT promoter region were al so much higher than that of wild-type, even though the 5’exon/enhancer region showed similar levels. This suggests the presence of a repressive element in the native core LAT promoter that might pr event increased transcriptional permissiveness of the LAT region at inappropria te times, such as during lytic infection. Additionally, because LAT promoter deletion mutant s are severely deficien t in reactivation in the rabbit, the difference in chromatin profiles between the mutant and the wild-type virus may implicate a requirement for establishment of a certain chromatin profile for normal reactivation and also suggests that deletion of the LAT pr omoter causes a loss of a regulatory element required for wild-t ype reactivation. When the chromatin profiles of wild-t ype HSV-1 and LAT promoter mutant, 17 Pst, were assessed following adrenergic induction of reactiva tion in the rabbit ocular model, no change in

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86 chromatin was evident for either virus be tween 0, 1, 2, and 4 hours post-induction, times corresponding to those tested in the mouse explant experiments. This finding suggests that chromatin remodeling is not criti cal to LAT-dependent reactivati on or that chromatin remodeling in response to mouse DRG explant is a much faster process than what occurs in the rabbit. For example, since the epinephrine is administered to the eye and reactivation occurs in the ganglia, it might take some time for the epinephrine to re ach and stimulate all cells in the ganglia, while in explant-induced reactivation, a uniform stressor (ganglion re moval from the animal) might stimulate cells to reactivate very rapidly. The deletion of the core LAT promoter in the 17 Pst mutant is 202-bp in size. This suggests that a number of binding sites and cis -elements were removed, some of which could normally contribute to cont rolling the level of transc riptional permissiveness. LAT Regulation through Promoter Function To investigate a possible regulator of th e LAT promoter, a cAMP response element, located 83 nucleotides upstream of the LAT transc riptional start site (-83CRE) was examined. A recombinant virus with mutation of the -83CRE wa s created and analyzed for alterations to the acute, latent, and reactivation phases of the vi ral lifecycle. The -83CRE recombinant was ultimately found to contain a second mutation, wh ich may have been a compensatory mutation that allowed an otherwise unviable recombinant to replicate. During acute infection, the mutant virus displayed attenuated virulence in mice that we re infected via the foot pad, and in cell culture the virus showed increased replic ation in fibroblasts and decreased replication in neuronal cells, suggesting a cell-type speci fic effect of the -83CRE site (or the compensatory mutation) in viral replication. The -83CRE recombinant virus was able to establish latency, express LAT, and reactivate. Interestingly, during latency in mi ce, the -83CRE mutant virus displayed decreased transcriptional permissiveness at the LAT promoter relative to the wild-type virus. This is in

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87 contrast to the findings for 17 Pst in the rabbit, in which de letion of the entire promoter (including the -83CRE site) caused dramatically in creased transcriptional permissiveness. Thus, the wild-type -83CRE may be important in regulation of the LAT promoter, probably by interacting with another nearby element as well as cellular factors, to maintain the configuration of the LAT region in a reactivation-compatible state. Removal of the -83CRE causes a decreased transcriptional permi ssiveness of the LAT promoter ye t the recombinant virus still reactivates from latency in the rabbit. Since th ere is another mutation in the recombinant, that second site may contribute to the chromatin configuration. For example, if ICP0 is mutated to a more active form, control of latency might not be as tightly controlled as in the wild-type virus; thus, the -83CRE mutant viru s can still reactivate because it is less repressed. Future experiments should address these questions. Findings suggest that the -83CRE is important in acute phase regulation of HSV-1, likely in concert with some other element(s) of th e genome. If the wild-type -83CRE binds a repressive protein to control LAT expression du ring the lytic infection, deletion of the element would cause misregulation of the LAT, potentially interfering with ICP0 transcription. If this occurred during the construction phase of a muta nt -83CRE virus, a second mutation in ICP0 may have occurred to overcome the problem. Charac terization of the second mu tation in the -83CRE recombinant is ongoing and should provid e information about interplay between the LAT promoter and other elements im portant to the viral life cycle. A Model for Reactivation A model has been proposed for steroid receptor function in which random and transient interactions between various fact ors and a promoter might not actu ally result in transcription; instead, since those events may be part of a se quential process, in which promoter modification and secondary recruitment of other factors ultima tely lead to transcri ption, ChIP experiments

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88 would indicate only some of the events in a popul ation at a particular tim e without giving a sense of the dynamic processes occurring in single cell s (reviewed in Hager et al., 2006). In other words, the binding of receptor complexes, and proba bly other transcription f actors, is a cyclical process; protein-DNA binding occurs without being stable and longterm, but the end result is usually promoter activation. S upport for this come s from experiments performed on single living cells using UV laser cro sslinking technology to monito r chromatin remodeling at a promoter; these studies indicated cycle times were less than a minute (reviewed in Hager et al., 2006). The approaches used in the experiments pe rformed in this dissertation used ChIP, thereby limiting observations to events occurring in th e collective group of cells in a ganglion. Perhaps the regulation of HSV reactivation coul d be modeled like the dynamic process that regulates steroid receptor activat ion, and at any given time, singl e “latently-infe cted” cells are actually producing lytic viral transcripts. Support for this comes from a study performed by Feldman et al. (2002), in which ganglia fr om latently-infected mice were assayed by in situ hybridization for the presence of lytic gene e xpression and viral replic ation. Assessment of thousands of neurons led to the finding that a small number of indivi dual neurons actually express lytic viral genes and replicate viral DNA, even though the ganglia as a whole is considered latently-infected. The authors also found indication that the host inflammatory response may prevent the stray neurons from yiel ding infectious virus (Feldman et al., 2002). Perhaps true reactivation does not actually occur until a group of those cells is synchronously transcribing lytic genes, and enough virus is produced to be infec tious. Activation of the cAMP pathway, or another stress-inducib le pathway, by a stress stimulus may be sufficient to not only trigger the synchronous initiation of reactivation in a larger number of cells, but also to create the

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89 correct environment (i.e., temporary depressi on of the local host response) for efficient production of abundant virus. The entire process of establishment of and r eactivation from latency is clearly complex. Findings can vary from system to system and viral strain to viral strain, possibly due to different types of reactivation. If regul ation at the level of chromatin is important, maybe a specific chromatin configuration must be established that is compatible with reactivation. If cis elements in the LAT rcr region are important, maybe timing and sync hronicity are critical for reactivation. However, it may be possible that both of these, as well as other regulatory mechanisms, play a role in preventing aberrant tran scription of lytic genes and co ntrolling reactivation. HSV-1’s ability to persist indefinitely in a host, unde tected by the immune system, and periodically reactivate to spread to a new host suggests a ve ry tightly regulated system that may include several ways for reactivation to occur. Work in the field of latency and reactivation will undoubtedly provide interesting and numerous pieces of the puzzle for years to come.

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90 APPENDIX REAL-TIME PCR PRIMER/PROBE SEQUENCES Table A-1. Real-time PCR primer/probes. Target Sequence (5’ to 3’) LAT Promoter (NC_001806: 118,263–118,323) Forward—CAA TAA CAA CCC CAA CGG AAA GC Reverse—TCC ACT TCC CGT CCT TCC AT Probe—TCC CCT CGG TTG TTC C LAT 5’ Exon (NC_001806: 119,326–119,397) Forward—GGC TCC ATC GCC TTT CCT Reverse—AAG GGA GGG AGG AGG GTA CTG Probe—TCT CGC TTC TCC CC ICP0 Promoter (NC_001806: 124,494–124,578) Forward—CCG CCG ACG CAA CAG Reverse—GTT CCG GGT ATG GTA ATG AGT TTC T Probe—CTT CCC GCC TTC CC ICP27/UL54 (NC_001806: 113,945–114,034) Forward—GCC CGT CTC GTC CAG AAG Reverse—GCG CTG GTT GAG GAT CGT T Probe—CAG CAC CCA GAC GCC ICP4 (NC_001806: 147,941–148,025 ) Forward—GAC GGG CCG CTT CAC Reverse—GCG ATA GCG CGC GTA GA Probe—CCG ACG CGA CCT CC HSV polymerase (NC_001806: 65,801–65,953) Forward—AGA GGG ACA TCC AGG ACT TTG T Reverse—CAG GCG CTT GTT GGT GTA C Probe—ACC GCC GAA CTG AGC A Rabbit GAPDH Forward—GCA CCA CCA ACT GCT TAG C Reverse—CCT CCA CAA TGC CGA AGT G Probe—CTG GCC AAG GTC ATC C Rabbit centromere Forward—GCT CCA GAA ACC TGA GAA AAC ATG AT Reverse—TGG AGA AAA GCG CAA TCT TCC T Probe—TTC GGC AAA TGC ATC CAA Mouse Xist (NR_001463: 857–925) Forward—GCT CTT AAA CTG AGT GGG TGT TCA Reverse—GTA TCA CGC AGA AGC CAT AAT GG Probe—ACG CGG GCT CTC CA Mouse APRT Forward—CTC AAG AAA TCT AAC CCC TGA CTC A Reverse—GCG GGA CAG GCT GAG A Probe—CCA GGG CCT CAC CAC C

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91 LIST OF REFERENCES Ackland-Berglund, C.E., Davido, D.J., Leib, D.A., 1995. The roles of the cAMP-response element and TATA box in expression of the herp es simplex virus type 1 latency-associated transcripts. Virology 210, 141–151. Ahmed, M., Lock, M., Miller, C.G., Fraser, N. W., 2002. Regions of the herpes simplex virus type 1 latency-associated transc ript that protect cells from apoptosis in vitro and protect neuronal cells in vivo. J. Virol. 76 (2), 717–729. Alberini, C.M., Ghirardi, M., Metz, R., Kandel, E.R., 1994. C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia. Cell 76 (6), 1099–1114. Amelio, A.L., Giordani, N.V., Kubat, N.J., O’Ne il, J.E., Bloom, D.C., 2006. Deacetylation of the Herpes Simplex Virus type 1 latency-associated transcript (LAT) enhancer and a decrease in LAT abundance precede an increase in IC P0 transcriptional permissiveness at early times postexplant. J. Virol. 80 (4), 2063–2068. Arias, J., Alberts, A.S., Brindl e, P., Claret, F.X., Smeal, T., Karin, M., Feramisco, J., Montminy, M., 1994. Activation of cAMP and mitogen re sponsive genes relies on a common nuclear factor. Nature 370 (6486), 226–229. Batchelor, A.H., Wilcox, K.W., O'Hare, P ., 1994. Binding and repression of the latencyassociated promoter of herpes simplex viru s by the immediate early 175K protein. J. Gen. Virol. 75 (4), 753–767. Becker, P.B., Hrz, W., 2002. ATP-dependent nu cleosome remodeling. Ann. Rev. Biochem. 71, 247–273. Bernstein, E., Hake, S.B., 2006. The nucleosome: a little variation goes a long way. Biochem. Cell Biol. 84 (4), 505–517. Bernstein, B.E., Humphrey, E.L., Erlich, R.L., Sc hneider, R., Bouman, P., Liu, J.S., Kouzarides, T., Schreiber, S.L., 2002. Methylation of hist one H3 lys 4 in coding regions of active genes. Proc. Natl. Acad. Sci. U.S.A. 99 (13), 8695–8700. Berthomme, H., Thomas, J., Texier, P., Epst ein, A., Feldman, L.T., 2001. Enhancer and longterm expression functions of herpes simplex vi rus type 1 latency-asso ciated promoter are both located in the same re gion. J. Virol. 75 (9), 4386–4393. Biswas, P.S., Rouse, B.T., 2005. Early events in HSV keratitis—setting th e stage for a blinding disease. Microbes Infect. 7 (4), 799–810. Bloom, D.C., Devi-Rao, G.B., Hill, J.M., Steven s, J.G., Wagner, E.K., 1994. Molecular analysis of herpes simplex virus type 1 during epinephr ine-induced reactivation of latently infected rabbits in vivo. J. Virol. 68 (3), 1283–1292.

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101 BIOGRAPHICAL SKETCH Nicole Giordani majored in microbiology at th e University of Arizona in Tucson, Arizona from August 1998 to December 2001. During this time, Nicole was part of the Undergraduate Biology Research Program (UBRP) and performed research on the alpha subunit of the RNA polymerase of Euglenoids under the supervision of Richard Hallick, Ph.D. After receiving her Bachelor of Science degree in December 2001, Nico le applied to graduate school and began the University of Florida’s Inte rdisciplinary Program (IDP) in August 2002. Upon earning her Ph.D., Nicole performed post-docto ral work in an academic setting.