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CHROMATIN AND COACTIVATORS IN HERPES SIMPLEX
VIRUS TYPE-1 GENE REGULATION

presented by

Sebla Bulent Kutluay

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CHROMATIN AND COACTIVATORS IN HERPES SIMPLEX VIRUS TYPE-1
GENE REGULATION

By

Sebla Bulent Kutluay

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Cell and Molecular Biology

2009

ABSTRACT

CHROMATIN AND COACTIVATORS IN HERPES SIMPLEX VIRUS TYPE-l
GENE REGULATION

By
Sebla Bulent Kutluay

The virion protein 16 (V P16) of HSV-1 serves as a prominent model for studying
transcriptional activation in eukaryotes. During lytic infection, HSV-l immediate early (IE) gene
expression is stimulated by the virion-borne transactivator protein VP16. In heterologous
expression systems, the VP16 activation domain (AD) can recruit various coactivators such as the
p300/CBP histone acetyltransferases (HATS) and the Em and Brg-l chromatin remodeling
complexes. Given prior findings that the HSV-l genome is mainly non-nucleosomal during lytic
infection, we hypothesized that such chromatin-modifying coactivators modify and remodel
nucleosomes on the viral genome enabling IE gene expression. We and others have shown that
during lytic infection the actively transcribed viral gene promoters and ORFs associate with
acetylated and methylated histone H3. Moreover, we have shown that the p300, CBP, Em and
Brg-l were recruited to [B gene promoters in a manner largely dependent on the VP16 AD.
Therefore, we hypothesized that the coactivators that are recruited by VP16 directly contribute to

IE gene expression.

This dissertation describes my efforts to elucidate whether the transcriptional
coactivators that are recruited by VP16 are important for viral gene expression and to
understand the mechanism behind histone depletion from the HSV-l genome. Disruption
of the expression of p300, CBP, PCAF and GCNS HATS by RNA interference did not
reduce IE gene expression during lytic infection. These results were supported by our

findings that IE gene expression was not impaired in mutant cell lines that did not express

functional coactivators. These results suggested that transcriptional coactivators are not

important for IE gene expression.

Given that coactivators are not required for IE gene expression, and that the viral genome
is depleted of histones during lytic infection, we have analyzed possible mechanisms of histone
depletion from the viral genome during various stages of lytic infection. We specifically asked
whether there is a role for VP16 AD, transcription per se or IE proteins in this process. To
address this question, we employed chromatin immunoprecipitation (ChIP) assays to detect the
presence of all four core histones on different regions of the viral genome during different stages
of lytic infection. These studies have indicated that VP16 and RNA Polymerase II (RNAP H)
contribute to histone depletion from IE promoters and coding regions, and that IE proteins are
also involved in preventing histone deposition at later stages of infection from other regions of

the viral genome.

Overall, we conclude that the HSV-l genome stays free of nucleosomes by the
action of VP] 6, RNAP II and IE proteins during lytic infection. Given that transcriptional
coactivators are not required for viral gene expression during lytic infection, we are
proposing that histones may be prevented from being deposited on the viral genome,
rather than being deposited and then removed fi'om the viral genome. Our ChIP results
also support this model. As such, the low amount of histones present on the viral genome
during lytic infection may not matter for the outcome of infection. Future research will
focus on the detailed mechanism that keeps the histones off the viral genome, and the
mechanism of how VP16 mediates transcription from nucleosomal templates in viva. It
will also be important to establish model systems where the necessity for coactivators in

reactivation from latency can be tested effectively.

This dissertation is dedicated to my family.

iv

ACKNOWLEDGEMENTS

I would like to thank my mentor, Steven J. Triezenberg, for teaching me how to become
an independent scientist. I appreciate his dedication and enthusiasm for science and his
unending patience in mentoring. I will fondly remember my time as a graduate student in

his lab.

I would also like to thank my thesis committee members, Andrea Amalfitano, Bill Henry,
Michele Pluck and Richard Schwartz for their support and assistance over the last five

years at MSU.

I would also like to thank all of the past and present members of the Triezenberg
laboratory. In particular, I would like to thank Francisco Herrera, Dean Shooltz and
Kanchan Pavangadkar for their advice and help in the early days of my graduate training.
I am grateful to James Doroghazi, Martha Roemer, Sarah DeVos and Jennifer Klomp for
their direct contributions to this dissertation. I would like to thank Glen Alberts and Xu

Lu for their advice and guidance in the lab.

I am grateful to my family overseas for supporting and encouraging me throughout my
graduate career. I value their understanding of the difficult times I had to go through over
the last five years. I would also like to thank to my friends of all nationalities at MSU and

VARI for making graduate school a colorful and memorable experience.

TABLE OF CONTENTS

List of Tables .......................................... , ....................................................................... viii
List of Figures ................................................................................................................... ix
List of Abbreviations ........................................................................................................ xi

Chapter I: Literature review

1.

5”

Chapter II:

SP‘MPP‘NI‘

Introduction ...................................................................................................... 2
1.1. Transcription in eukaryotes ...................................................................... 3
1.1.1 Histone modifications and transcription ................................... 4
1.1.2 Chromatin remodeling and transcription .................................. 5
1.1.3 Incorporation of histone variants and role of histone
chaperones in transcription ................................................................ 6
1.1.4 Nucleosome removal during transcription ............................... 8
1.2. Chromatin during herpesvirus infections .................................................. 9
1.2.1 Chromatin on the HSV-l genome during lytic infection ....... 12
1.2.2 Chromatin during HSV—l latency .......................................... 20
1.2.3 Chromatin during the infection of other herpesviruses .......... 25
Perspectives .................................................................................................... 3 1
Acknowledgements ........................................................................................ 34
References ...................................................................................................... 35
Role of transcriptional coactivators in HSV-l infection
Abstract .......................................................................................................... 54
Introduction .................................................................................................... 56
Methods .......................................................................................................... 60
Results ............................................................................................................ 64
Discussion..................................... ................................................................. 89
Acknowledgements ........................................................................................ 97
References ...................................................................................................... 98

Chapter III: Curcumin inhibits HSV-l infection by a mechanism independent of
p300/CBP HAT activity

NP‘P‘PP’P?‘

Abstract ........................................................................................................ 1 08
Introduction .................................................................................................. 1 09
Methods ........................................................................................................ l 1 2
Results .......................................................................................................... 1 16
Discussion .................................................................................................... l 3 1
Acknowledgements ...................................................................................... 1 35
References .................................................................................................... 1 36

vi

Chapter IV: Mechanisms of histone depletion from the viral genome

1. Abstract ........................................................................................................ 142
2. Introduction .................................................................................................. 143
3. Methods ........................................................................................................ 148
4. Results .......................................................................................................... 151
5. Discussion .................................................................................................... 175
6. Acknowledgements ...................................................................................... 181
7. References .................................................................................................... 182

Chapter V: Synthesis and future directions
1. Potential mechanisms leading to the lack of histones on the HSV-l genome

during lytic infection .................................................................................... l 88
2. Transcriptional coactivators and HSV—l infection ...................................... 194
3. Role of histone chaperones in HSV—l infections ......................................... 197
4. Concluding Remarks .................................................................................... 198
5. References .................................................................................................... 199

vii

LIST OF TABLES

Table l: siRNA sequences and catalogue numbers used in this study .......................... 105

Table 2: Oligonucleotides used as primers in quantitative real-time PCR .................... 106

viii

LIST OF FIGURES

Chapter II:

Figure 2.1: Disruption of p300 and CBP expression by RNAi does not decrease
HSV-l IE gene expression ................................................................................... 67

Figure 2.2: HSV-l IE gene expression is not impaired in SiHa cells but
augmented in the presence of wild-type p300 ..................................................... 70

Figure 2.3: RNAi of PCAF and GCNS does not decrease HSV-l IE
gene expression .................................................................................................... 73

Figure 2.4: Disruption of BRM and Brg-l expression does not decrease HSV-l IE
gene expression .................................................................................................... 77

Figure 2.5: HSV-l IE gene expression in SW13 and C33—A cells that do not
express BRM and Brg-l remodeling enzymes .................................................... 81

Figure 2.6: HSV-2 superinfection induces IE gene expression in
RPS-infected cells ................................................................................................ 85

Figure 2.7: Coactivators are not required for HSV-2-dependent induction of IE
gene expression from RPS genomes .................................................................... 87

Figure 2.8: H3K18 but not H3K14 acetylation is reduced when p300 and CBP

expression is disrupted together ........................................................................... 93
Chapter III:

Figure 3.1: Effects of curcumin on HSV-l plaque formation and viral

replication .......................................................................................................... 1 18

Figure 3.2: Effects of curcumin on HSV-l IE gene expression ........................ 121

Figure 3.3: Curcumin decreases occupancy of RNA polymerase II but not of
VP16 at IE genes ................................................................................................ 123

Figure 3.4: Curcumin does not affect histone H3, p300, or CBP occupancy, nor
H3 acetylation, at IE genes ................................................................................ 127

Figure 3.5: Knocking down p300 expression does not affect HSV-l IE gene
expression .......................................................................................................... 1 30

ix

Chapter IV:

Figure 4.1: Similar numbers of RPS and KOS viruses enter HeLa cells and RPS
viruses are debilitated in replication .................................................................. 152

Figure 4.2: Core histone occupancy on the viral genome increases in RPS lytic
infections ............................................................................................................ 155

Figure 4.3: Inhibition of transcription by actinomycin D increases the core histone
occupancy on the HSV—l genome ...................................................................... 159

Figure 4.4: Changes in histone occupancy on the HSV—l genome upon inhibition
of IE protein synthesis by cycloheximide .......................................................... 163

Figure 4.5: HSV—l IE gene expression is induced by HSV-2 superinfection
independent of de novo protein synthesis ............................................... ., ......... 167

Figure 4.6: HSV-2 superinfection does not cause major changes in histone
occupancy or covalent histone modifications on the RPS genome .................... 168

Figure 4.7: Histone and RNAP II co-occupancy on RPS IE genes upon HSV-2
superinfection ..................................................................................................... 1 73

ChIP

DE

HAT
HSV-1
HSV—2
IE

L

RNAP II
Seq-ChIP
VP16

VP16 AD

LIST OF ABBREVIATIONS

Chromatin immunoprecipitation

Delayed early

Histone acetyltransferase

Herpes simplex virus type-1

Herpes simplex virus type-2

Immediate early

Late

RNA polymerase II

Sequential chromatin immunoprecipitation
Virion protein 16 from herpes simplex virus

Activation domain of VP16

xi

CHAPTER ONE

Kutluay, Sebla, B., Triezenberg, Steven, J. (2009). Role of Chromatin During
Herpesvirus Infections. BBA General Subiects.

Chapter 1
LITERATURE REVIEW
1. INTRODUCTION

The last two decades have witnessed exciting developments in our knowledge of
how eukaryotic gene transcription is regulated. Early developments focused on the cis-
regulatory elements associated with specific gene promoters and on the trans-acting
factors that bind to these elements. More recent progress has revealed dynamic aspects of
chromatin structure and the mechanisms whereby chromatin and its modifications

influence gene expression.

DNA viruses have long served as model systems to elucidate various aspects of
eukaryotic gene regulation, due to their case of manipulation and relatively low
complexity of their genomes. In some cases, these viruses have revealed mechanisms that
subsequently are recognized to also apply to cellular genes. In other cases, viruses adopt
mechanisms that prove to be exceptions to the more general rules. The double-stranded
DNA viruses that replicate in the eukaryotic nucleus typically utilize the host cell RNA
polymerase II (RNAP II) for viral gene expression. As a consequence, these viruses must
reckon with the impact of chromatin on active transcription and replication. Unlike the
small DNA tumor viruses, such as polyomaviruses and papillomaviruses, the relatively
large genomes of herpesviruses are not assembled into nucleosomes in the virion and stay

predominantly free of histones during lytic infection. In contrast, during latency, the
herpesvirus genomes associate with histones and become nucleosomal, suggesting that

regulation of chromatin per se may play a role in the switch between the two (stages of

infection, the exact mechanism of which is a long-standing puzzle in the biology of

herpesviruses.

In this review we will focus on how chromatin formation on the herpes simplex
type-1 (HSV—l) genome is regulated, citing evidence supporting the hypothesis that the
switch between the lytic and latent stages of HSV-1 infection correlates with changes in
the chromatin state of the HSV—l. Before going into the details of HSV—1, we will briefly
summarize some of the recent advancements in regulation of chromatin and transcription

by RNAP II as it pertains to the rest of this review.

1.1. Transcription in eukaryotes:

Eukaryotic DNA is packaged in the form of nucleosomes, whereby approximately
147 bp of DNA is wrapped around a protein octamer that consists of two copies of each
core histone (H2A, H2B, H3 and H4). Further compaction of nucleosomes is mediated by
the linker histone H1 and other non-histone proteins. Although RNAP II can transcribe
efficiently in vitro from naked DNA templates, the packaging of DNA into nucleosomes
inhibits transcription. The past few decades have witnessed great progress in our
understanding of how the inhibitory effect of chromatin on transcription can be
overcome. Four principal mechanisms include the covalent modification of histone tails
and globular domains, remodeling of nucleosomes, incorporation of histone variants, and
removal or disruption of nucleosomes at actively transcribed genes. These four general
mechanisms will be described briefly before turning to the role of chromatin and its

modification during herpesvirus infections.

1.1.1. Histone modifications and transcription

Covalent or post-translational modifications of the amino-terminal tails of core
histones have been extensively characterized (84), although the globular domains can
also be modified (124, 141 , 178, 183, 186). The most prominent covalent histone
modifications include acetylation, methylation, ubiquitinylation, phosphorylation, and
prolyl isomerization (84). In many cases, modifications on specific residues of particular
histones have been correlated as either positive or negative markers of transcriptional
activity (84). Genome-wide studies that employed chromatin immunoprecipitation (ChIP)
assays coupled with DNA microarrays or high-throughput sequencing have shown that
particular modifications are predominantly localized to distinct regions of target genes,
such as the upstream regulatory regions, core promoters, or the 5' and 3' portions of the
transcribed regions (144). For instance, histone H3 acetylated on lysines 9 and 14
(H3K9/K14ac) localizes to the promoter and 5' ends of actively transcribed genes.
Methylation of histone H3 can be an indicator of either active or inactive transcription,
depending on which lysine residue is modified. Histone H3 methylation also follows a
distinct pattern of localization through the body of a gene; for example, H3K4me3 is
mainly present around the transcriptional start site, whereas H3K36me3 is localized
towards the middle and 3' ends of actively transcribed genes. Other H3 methylation
marks, such as H3K9me3 or H3K27me3, are strictly associated with inactive

transcription and are observed over broad regions of silenced genes.

In parallel with the identification of covalent histone tail modifications has come
the discovery of the corresponding enzymes that catalyze these reactions. For instance,

histone methyltransferases are rather specific for the target lysine or arginine residue.

4

Histone acetylation is somewhat less specific: a given histone acetyltransferase (HAT)
might modify several residues, and several different HATS might have overlapping
substrate specificities. For instance, the HATS p300, CBP, and PCAF can all acetylate
H3K14 (105, 115, 158). As a rule, the covalent marks are reversible by enzymes such as
histone deacetylases (HDACs) and lysine demethylases, indicative of the highly dynamic
nature of chromatin modifications and multiple potential levels of trancriptional

regulation (84).

Covalent modifications of histones are thought to have two principal
consequences. The first is the direct impact of modification on higher-order chromatin
structure. For instance, the loss of positive charge on lysines upon acetylation is
associated with relaxed chromatin structure (84). The second potential outcome is the
recognition of specific histone modifications by other proteins that function as
transcription factors or coactivators. Two examples of such mechanism are proteins
containing bromodomains, which bind to acetylated lysines, and proteins containing
chromodomains, which bind to methylated lysines (25, 161). Since a number of bromo-
or chromodomain-containing proteins are themselves chromatin-modifying enzymes, this
recognition enables the propagation or cooperativity of histone modifications and

chromatin remodeling (25, 161).

1.1.2. Chromatin remodeling and transcription

The second major class of chromatin-modifying factors comprises protein
complexes that utilize ATP hydrolysis to induce changes in the positions of nucleosomes

on DNA and hence are called chromatin-remodeling complexes. Chromatin remodeling

may result in sliding of the nucleosomes on DNA, DNA looping on the nucleosome

particle, or histone octamer transfer in trans (40, 154).

Several families of chromatin remodeling complexes have been identified. The
prototypes of these families include SWI/SNF, ISWI, INO80, and NURD/Mi-2/CHD, all
of which contain an ATPase subunit and have both similar and distinct functions. For
instance, the ISWI and NURD/Mi-2/CHD families are both involved in transcriptional
repression, yet a separate function of the ISWI family is to induce ordered chromatin
assembly. The SWI/SNF family, on the other hand, is primarily associated with active
transcription. In mammals, remodelers of the SWI/SNF family are represented by two
separate complexes that have hBRM and BRGl as their ATPase subunits. In addition to
their role in transcription, hBRM and BRGl remodeling complexes in mammals are

involved in processes such as cancer progression, differentiation, and development (154).

1.1.3. Incorporation of histone variants and role of histone chaperones in

transcription

The third mechanism that influences the impact of chromatin on gene regulation
is the incorporation of histone variants. Whereas the canonical core histones are each
encoded by multiple genes that are expressed predominantly in the S phase of the cell
cycle, histone variants are encoded by single-copy genes that are expressed independent
of DNA replication. Histone variants are thought to exert their actions mainly by
influencing the stability of nucleosomes or higher-order chromatin structure, but not by

differential covalent modifications, as in most cases the sites for covalent histone

modifications are conserved among the variants. Another theory postulates that exposure

of different surface residues in histone variants may serve as binding sites for other

proteins (70, 72).

Although H1, H2A, and H3 have multiple variants, no histone variants have been
identified for H2B and H4. The H2A variants in humans include H2A.X, H2A.Z, H2A-
Bbd, and macroH2A, each of which has a distinct localization pattern and function (70,
72). For instance, macroH2A is localized to the inactive X chromosome, where it is
thought to contribute to heterochromatin formation. In contrast, H2A-Bbd is excluded
from the inactive X chromosome and accumulates at actively-transcribed genes. H2A-
Bbd is an exceptional histone variant in that it shares only 48% sequence identity with
histone H2A and lacks a number of structural features characteristic of histone H2A
family. As such, this histone variant is thought to participate in destabilization of
nucleosomes, which then facilitates the recruitment of transcription factors and
coactivators that facilitate active transcription (47).Although in yeast H2A.Z prevents the
spread of heterochromatin, in higher eukaryotes it might also function in the formation of
heterochromatin. The principal function of H2A.X is not in transcription but in DNA
repair: phosphorylated H2A.X (y-H2A.X) marks the regions of double-stranded DNA
breaks and thus aids in recruiting the DNA repair machinery. Among the two major H3
Variants, CENP-A is localized exclusively to the centromeres and contributes to
f(>1‘rnation of kinetochore and chromosome segregation. The other histone H3 variant,
H 3 - 3, differs from the canonical H3 by only a few amino acid substitutions but is
expressed throughout the cell cycle and is present in transcriptionally active regions. A
l arge number of histone H1 variants, which share a conserved core domain yet have more

di Vergent N- and C-temini, have been identified in humans. Although histone H1 variants

were initially thought to have redundant functions, recent findings indicate that they may

also have specific roles in gene regulation (67).

The assembly of histones and histone variants into nucleosomes requires the
activities of a number of proteins and protein complexes (59, 156). CAF l and HIRA are
two such assembly factors that incorporate H3.] (canonical histone H3) and H33 into
nucleosomes in a replication-dependent and -independent manner, respectively. Another
histone chaperone, Asfl a, interacts with both CAF 1 and HIRA, and as such it is involved
in both replication-dependent and -independent histone assembly. Assembly of other
histone variants might also be mediated by specific protein complexes, most of which are

yet to be identified. For instance the SWRl complex, which contains SWI/SNF-type

remodeling activity, is involved in deposition of histone variant H2A.Z in yeast (119).

Other histone chaperones interact with RNAP II and contribute to overcoming the
nucleosomal barrier to transcription. Histone chaperone NAPl preferentially interacts
with H2A-H2B dimers and mediates histone shuttling between nucleus and cytoplasm, as

well as the removal of H2A-HZB dimers during transcription, which may allow further
removal of histones and enable the passage of RNAP 11. Two other chaperone-like
factors, FACT and Spt6, remove and reassemble histones during elongation by RNAP II,
enabling the passage of RNAP 11 through nucleosomes while maintaining the structure of

Chromatin and inhibiting cryptic transcripts (6, 73, 146).
1.1.4. Nucleosome removal during transcription

A fourth mechanism at work that might contribute to active transcription is the

partial or complete depletion of nucleosomes from gene promoters or transcribed regions

(61, 94, 187). The initial evidence that nucleosome structure is disrupted during
transcription by RNAP 11 came from in vitro studies, where the absence of an H2A-H2B
dimer from nucleosomes both increased the affinity for and stimulated transcription by
RNAP II (48, 49). In accord with these initial observations, recent in vitro evidence
indicated that passage of RNAP 11 leads to the elimination of a H2A-H2B dimer fiom
nucleosomes (78, 183). In support of these in vitro assays are observations of increased
rates of histone exchange that correlated with transcriptional activity in yeast (68) and in
Physarum (174). Histone chaperones and chaperone-like proteins, such as FACT and

Spt6 that associate with the elongating RNAP II are likely participants in this process.

Whereas H2A-H2B dimers are depleted from DNA regions traversed by RNAP
II, promoters of actively transcribed or transcriptionally competent yeast genes are often
relatively fi'ee of nucleosomes (32, 195). Moreover, histone octamers can also be lost
throughout the coding regions of highly transcribed regions in yeast (83, 90, 98, 147,
159), although another study indicated the contrary (199). Hi gh-throughput genome-wide
screens have also shown that high rates of histone turnover within coding regions, but not
promoters, correlate with RNAP II density (28). These findings together lead to the
conclusion that active transcription by RNAP II correlates with the partial disruption or

removal of nucleosomes.
1.2. Chromatin During Herpesvirus Infections

Herpesviruses vary greatly with respect to the cell types infected and the clinical
diseases they cause, yet they share common structural features. A typical herpesvirus

Virion contains a linear double-stranded DNA of 120-230 kilobase-pairs packaged in an

icosadeltahedral capsid. The capsid is surrounded by an amorphous protein coat, known
as the tegument, and a lipid envelope in which viral glycoproteins are embedded.
Common to all herpesviruses is the establishment of life-long latent infections after a
phase of lytic infection. Reactivation from latency in response to any of a number of
stresses results in recurrent infections. The mechanisms by which herpesviruses establish
latency and reactivate remain unresolved, although various mechanisms have been
proposed in recent years. In this review, we will primarily focus on the regulation of
chromatin and chromatin modifications during the lytic and latent stages of a model
herpesvirus, herpes Simplex virus type 1 (HSV-l , also known as human herpesvirus 1 or
HHV-l ), as it presents a potential mechanism for the switch between the two stages of

infection.

HSV—l belongs to the Alphaherpesvirinae subfamily together with the human
viruses herpes simplex virus type 2 (HSV-2 or HHV-Z) and varicella zoster virus (VZV
or HHV-3), and a number of viruses that infect various animal species. HSV-l
commonly causes oral cold sores but can also cause corneal infections and encephalitis.
By adulthood, most of the world population becomes seropositive for HSV-l , yet not all

of those individuals present symptomatic infection.

The life cycle of HSV-1 is characterized by an initial phase of lytic infection in
epithelial cells, followed by a latent phase in the neurons of the trigeminal ganglion.
During the lytic phase, attachment of the virus to the host cell membrane by interaction of
Viral glycoproteins with cellular receptors leads to membrane fusion and the release of
nuCleocapsid and tegument components to the cytoplasm. The viral capsid is then

transported to the nuclear pores, through which the viral genome is released into the

10

 

nucleus. The major viral transcriptional activator protein, VP16, which is one of the
tegument proteins, is also transported to the nucleus by mechanisms yet undefined. VP16
forms a complex with two cellular proteins, Oct-1 and HCF-l, and binds to specific cis-
acting sequences in the promoters of immediate early (IE) genes to stimulate their
transcription (189). Expression of delayed early (DE) and late (L) genes is, in turn,

dependent on some of the IE proteins, such as ICP4 (140, 186).

Following the release of infectious virions from epithelial cells at the primary site
of infection, some HSV-l virions infect the surrounding sensory neurons. The viral
nucleocapsid is transported via retrograde axonal transport to the cell bodies of the
neurons in the trigeminal ganglion, where HSV-l establishes latency. During the latent
phase of infection, the viral genome is maintained as a circular episome and viral gene
expression is repressed, with the exception of the latency—associated transcript gene
(LAT), which is the only gene continuously transcribed during latency. Stress stimuli,
such as UV exposure or thermal injury, lead to reactivation of the virus by an unknown
mechanism. HSV—l then travels through the sensory neurons by anterograde transport
and causes recurrent infections in the epithelial cells, usually at the same location as the

primary infection.

Several related aspects regarding the role of chromatin in the context of HSV-1
infection have drawn significant attention in recent years. One question is how the viral
genome remains substantially non-nucleosomal during lytic infection. Another issue is
the mechanism of the transition from lytic infection, in which the viral genomes are
Predominantly histone-free, to latency, in which viral genomes are nucleosomal. A third

QUeStion seeks the molecular mechanism by which the viral genomes are released from

11

 

the inhibitory effects of chromatin during reactivation from latency. We and others have
proposed that the regulation of chromatin on the viral genome itself may be a determining
factor in the switch between lytic and latent infections. In this section, we will focus on
some of the recent developments on this subject. For further insight, see the excellent

recent review by Knipe and Cliffe (80).
1.2.1. Chromatin on the HS V-I genome during lytic infection

Unlike the small DNA tumor viruses of the polyomavirus and papillomavirus
families, the genome of HSV-1 is not packaged with histones in the virion particle (18,
133, 136); instead, the polyamine spermine provides the counterions for the phosphates
of viral DNA (45). A number of years ago, nuclease-digestion studies indicated that the
viral DNA remains predominantly free of nucleosomes throughout the lytic infection
(100, 101, 121). Moreover, histones are excluded from viral replication compartments of
infected cell nuclei (120, 163), and GFP-tagged linker histone variants in infected cells
have a higher mobility assessed by fluorescence recovery after photobleaching (FRAP)
assays (20). More recently, ChIP assays employed by several groups have indicated that
histones (typically represented by histone H3) are present on the viral genome at much
lower levels than on cellular genes; that is, the fiaction of input DNA that is
immunoprecipitated by anti-histone antibodies is much lower for viral DNA than for
cellular DNA present in the same sample (63, 65). The low levels of histones on the viral
genome can be interpreted in several ways. The first possibility is that most viral
genomes carry a few randomly-distributed histones throughout the viral genome.
Alternatively, chromatin might form on a small fraction of viral genomes (perhaps as an

innate defense mechanism against foreign DNA), whereas the rest of transcriptionally

12

 

active viral genomes remain free of histones. A third possibility is that histone deposition
on a small fraction of viral genomes might be a requirement for engaging those genomes
in the transcriptional activation mechanisms typically employed for host genes.
Observations that histone modifications associated with active transcription, such as
H3K4me3 and H3K9/Kl4ac, are found on the viral genome during lytic infection (63, 65,

76) would be consistent with the first or third models.

Studies probing the possible mechanisms of histone depletion from and histone
modifications on the viral genome during lytic infection have focused on recruitment or
displacement of chromatin-modifying coactivators by viral or cellular regulatory proteins.

The following sections will summarize some of these recent findings.
Role for VP16:

VP16 is the main transcriptional activator of IE genes and has served as a model
transcriptional activator for decades, often in artificial experimental settings in which the
VP16 transcriptional activation domain (VP16 AD) is fused to a heterologous DNA-
binding domain such as that from the yeast Gal4 protein (151). In both in vitro
experiments and in transformed yeast or transfected mammalian cells, the VP16 AD can
interact with basal transcription factors such as TFIID, TFIIA, TFIIB, and TFIIH (7, 62).
The VP16 AD can also interact with a number of transcriptional coactivators that

POtentiate transcription and can recruit these coactivators to promoters of target genes.
Some of these coactivators include p300/CBP HATS (8, 57, 178, 184), PCAF and GCNS
HATS (57, 178, 180, 184), and SWI/SNF remodeling complexes (53, 125, 126, 196).

Interactions of VP16 AD with coactivators may induce decondensation of a highly

13

compact 90 Mbp heterochromatic amplified chromosome region, independent of
transcriptional activity (12). A similar study also suggested that chromatin
decondensation mediated by VP 1 6 AD is not localized but rather propagates over larger
regions (>100 kbp) (177). Recruitment of SWI/SNF by VP16 AD also leads to eviction
of histone octamer from reconstituted mononucleosomes and nucleosome arrays in vitro
(53). These findings all point to an attractive model in which VP16 recruits chromatin-
modifying coactivators in order to regulate chromatin formation on the HSV-l genome

during different stages of infection.

We have tested certain aspects of this model during HSV-l lytic infection using a
mutant virus strain (designated RPS) that lacks sequences encoding the VP16 AD. During
lytic infection by RPS, expression of IE genes was greatly impaired (168, 192), indicating
the crucial role of the VP 1 6 AD in initiating the viral gene expression cascade. Moreover,
RPS could not effectively establish latent infections in the central or peripheral nervous
system of immunocompetent mice (168). ChIP studies using cells infected with RPS and
or its wild-type parent strain, KOS, indicated that the HATS p3 00 and CBP and the
chromatin remodeling enzymes hBRM and Brg-l are recruited to HSV-l IE gene
promoters in a manner mostly dependent on the presence of the activation domain of
VP16 (63). Interestingly, p300 and CBP are also components of the ND10 structures,
which assemble on the incoming viral genomes and become replication compartments at
later stages of infection (97, 111, 118). Although ND10 structures are thought to be
inhibitory for viral gene expression, the presence of p300 and CBP in these structures

suggests that some components of ND10 may be beneficial for viral gene expression.

14

ChIP assays also indicated that histone H3 levels throughout the RPS genome were

higher than on wild-type viral genomes (63).

These observations are consistent with a model in which histone deposition on the
IE genes is reversed by the activities of chromatin-modifying coactivators recruited by
VP16. An extension of this model predicts that the functions of these coactivators might
be essential (or at least important) for effective activation of IE gene expression. This
prediction was tested by experiments in which the expression of particular coactivators
was disrupted by siRNAs prior to viral infection. Contrary to the predictions, such
siRNAs had little or no effect on viral IE gene expression, at either high or low
multiplicities of infection (95). Even when combinations of siRNAs were used to
circumvent potential redundancy among or between various classes of coactivators, viral
gene expression was not inhibited. Moreover, cell lines in which various coactivators are
absent or defective were fully capable of supporting viral gene expression (95). These
results indicate that the coactivators tested are not essential for VP16-mediated activation
of IE gene expression during lytic infection. The possibility that coactivators are required
during reactivation from latency, when the viral genome transitions from a nucleosomal
to a nucleosome-free state, is addressed more in detail in subsequent sections of this

review.
Role for HCF-l and histone mihvlation

The cellular protein HCF -1 has been known for some time as a component of the
Stable VP16-induced complex (VIC) on IE promoters during lytic infection (44, 87, 88,

189). Recent evidence suggests that HCF-l may also influence histone modifications on

15

HSV—l DNA. HCF-l can interact both with the Sin3 histone deacetylase complex and
with the Setl/Ash2 histone H3K4 methyltransferase complex (190), which are associated
with transcriptional repression and activation, respectively. Although it seems
contradictory that HCF -1 interacts with these complexes with different transcriptional
outcomes, VP16 selectively binds to HCF -1 that is associated with Setl/Ash2, but not
Sin3 (190). Consistent with this observation, promoters of several temporal classes of
HSV-1 genes were found to associate with H3K4me3 in lytically infected cells (65).
Although disruption of Setl expression by RNAi resulted in a decrease in H3 K4me3
levels on viral genes, the impact on IE gene expression was rather modest and evident
only at later times in infection (65). A role for HCF -1 and Setl was also suggested by
Narayanan et al. (124) for VZV IE gene expression. In that study, HCF-l was shown to
be required for the recruitment of Setl to the VZV IE62 promoter in transfection-based
assays and during lytic infection. Further studies are needed to clarify the importance of

Setl and histone methylation for viral IE gene expression.

Role for ICPO and ND10

ICPO is a multi-functional IE protein that may contribute to regulation of
chromatin on HSV-l DNA. Although ICPO does not directly bind to DNA, it stimulates
transcription from all kinetic classes of viral promoters (33, 42, 131, 132, 142). However,
absence of ICPO results in decreased viral gene expression only at low, but not at high,
multiplicities of infection (11, 15, 71, 150, 165). In addition, the requirement for ICPO is
dependent on the cell type; for example, ICPO is not required for productive infection in

UZOS osteosarcoma cells even at low MOIs (193).

16

One of the ways that ICPO may activate transcription is by stimulating the
degradation of the host PML protein, leading to disruption of ND10 structures (3 S).
ND10 structures form around the incoming viral DNA and are implicated in
transcriptional repression of viral genomes (38, 127). Disrupting the expression of ND] 0
components such as PML or Sp100 partially complemented a viral ICPO null mutation
(37, 38). In contrast, overexpression of PML or blocking the ICPO—mediated disruption of
ND10 structures had no inhibitory effect on viral gene expression (111). Transcriptional
coactivators such as p300 and CBP, which are associated with active transcription, also
colocalize with ND10 structures (97, 111, 118). Therefore, disruption of ND10 structures
by ICPO may not only relieve a general repression mechanism, but also may allow the
relocalization of factors that may positively regulate viral transcription. Interestingly, the
ICPO protein of bovine herpesvirus 1 can associate with p300 HAT (198), but it is not
clear if p300 is a partner for HSV-l ICPO in mediating the changes in chromatin structure

in the context of lytic infection.

ICPO may also prevent heterochromatin formation more directly by inhibiting the
activity of histone deacetylases (HDACS). ICPO interacts with several mammalian
HDACs (110) and forms a complex with the REST/CoREST/HDAC repressor complex,
leading to the dissociation of HDAC] from the complex (51, 52). Although HDAC
inhibitors, trichostatin A and sodium butyrate, increased viral gene expression during
infection by ICPO mutant viruses in some systems (138, 139), conflicting results were
obtained in the relatively non—permissive human fibroblasts, where trichostatin A had no
effect on the replication of ICPO mutant HSV-l (37). Interestingly, a recent study

indicated that the absence of ICPO correlated with an increase in histone H3 levels and a

17

decrease in the fraction of H3K9/Kl 8ac on the viral genome during lytic infection,
perhaps as a result of the increase in histone H3 occupancy on the viral genome rather
than a decrease in histone acetylation per se (17). It should also be noted that unlike
HDAC inhibitors which induce global changes in histone acetylation, ICPO does not
increase the acetylation of histone H4 (110). As such, whether HDACS contribute
directly to the silencing of viral genomes and whether an important function of ICPO is to

block HDACS to allow viral gene expression remains an open question.

ICPO also promotes the degradation of two histone H3 variants, the CENP-A and
CENP—C kinetochore proteins, thereby inducing mitotic arrest or abnormal cytokinesis
(34, 109). Whether this function of ICPO is important for the outcome of viral infection is
not yet known. Given that the viral genomes are nucleosomal during latent infection and
that ICPO may play a crucial role during reactivation, one attractive hypothesis is that
CENP-A and CENP-C associate with viral genomes during latent infection and that ICPO

has a requisite role in removing these proteins from the viral DNA during reactivation.

Other viral and cellular proteins:

 

The protein kinase encoded by the viral Us3 gene may also influence chromatin-
related events, based on evidence that the U83 kinase blocks the activity of HDAC1/2
(presumably by phosphorylation) and that the U53 kinase can enhance expression of a
reporter gene transduced into U208 cells (138, 139). However, the evidence connecting
the effects of Us3 on HDACS with the effect on gene expression is at present only

circumstantial.

l8

One of the DE proteins, the single-stranded DNA binding protein ICP8,
coprecipitates hBRM and Brg-l remodeling enzymes (172). The functional consequence
of this interaction during lytic infection is not yet well—defined. This association is a
useful reminder that the chromatin remodeling activity of hBRM and Brg-l may
contribute to both transcription and replication at different stages of infection by

depleting the histones from the viral genome.

Another mechanism that might conceivably prevent histone deposition on the
viral genome during lytic infection is the formation of viral “chromatin” comprising viral
proteins. The possibility that proteins other than cellular histones might associate with
the viral genome is not unprecedented. For instance, during spermatogenesis
conventional histones are replaced by protamines, which are rich in arginine and provide
very high levels of compaction (77). In addition, the core protein VII of adenoviruses,
which also replicate in the nucleus, associates with the viral genome throughout infection
(191). At later stages of HSV infection both ICP4 and ICP8 accumulate in viral
replication compartments (8]). At present, no quantitative data exist to Show whether
these proteins coat the viral genomes during lytic infection to an extent that might prevent
histone deposition. Moreover, given that both ICP4 and ICP8 are synthesized de novo in
infection, this hypothetical mechanism would not keep the viral genomes free of histones

at earlier stages of infection.

The preceding paragraphs discuss several viral proteins that modulate the cellular
chromatin and transcription machinery. In addition, however, the nuclear architecture of
the infected cell may influence heterochromatin formation on the viral genome. A recent

report indicated that absence of lamin A, a major structural component of the nuclear

l9

lamina, resulted in defects in viral gene expression and replication as well as a significant
increase in heterochromatin formation on the viral genome (162). This provides an
attractive model in which the localization of incoming viral genomes to specific regions
in the nucleus may inherently prevent heterochromatin formation on the viral genome and
provide an easy access to transcription machinery of the host. The details of such a
mechanism, including any involvement by the nuclear pore complexes through which

viral DNA enters the nucleus, remain fertile grounds for future investigation.

1.2.2. Chromatin during HS V-I latency

The release of HSV-1 from epithelial cells at the primary site of infection can lead
to subsequent infection of surrounding sensory neurons, with two potential outcomes.
Infection of some sensory neurons by HSV-1 may result in lytic infection (41 , 85, 181)
leading to cell death and clearance of these neurons fiom the trigeminal ganglia.
However, in another fraction of sensory neurons, latent viral infections are established in
which lytic gene expression is suppressed and the latency-associated transcript (LAT)
becomes the only viral gene that is continuously expressed (164). Splicing of the primary
8.3-kb LAT leads to the accumulation of two stable introns in the nucleus (39, 182).
Although some studies suggest that LAT encodes one or more polypeptides (30, 175), the
currently prevailing model asserts that LAT is not translated (31). One of the functions of
LAT is to reduce the expression of lytic genes during both acute (41) and latent infections
(16) in sensory neurons as well as in cultured neuronal cells (114). An exciting recent
development provides insight into how LAT may mediate the suppression of lytic genes.
Several microRNAs (miRNAs) were found to be encoded within the LAT primary

transcript of both HSV-l and HSV—2 (169, 170, 179). These miRNAs can down-regulate

20

IE gene expression in transfection-based assays (170, 179). Future work will address
whether point mutations in LAT that block the down-regulation of IE gene expression
indeed affect establishment of or reactivation from latency. LAT can also block apoptosis
in rabbit ganglia or when expressed ectopically in cultured cells (135), although the
mechanism is not yet fully defined. Absence of LAT correlates with increased productive
infection and cell death in neuronal cells, which might be explained by the effects of LAT
on lytic gene expression and apoptosis (176). For more insight on the functions of LAT,

we refer the readers to recent reviews (9, 80).

The promoter region of LAT shows neuronal specificity (5, 200) and contains a
TATA-box as well as regulatory elements about 700 bp upstream of the transcriptional
start site (29). In addition, an enhancer that is responsible for long-term LAT expression
maps downstream of the transcriptional start site (107, 108). Although the transcription
factors that bind to the LAT promoter have not yet been completely defined, potential
regulators include ATF/CREB (7S), STATl (86) and EGR (171). Recently, insulator-like
elements that are bound by CCCTC-binding factor (CTCF) were identified upstream of
the LAT promoter and in the LAT intron (3); these elements may contribute to regulation

of chromatin on the viral genome during latency, as explained in more detail below.
Histone modifications during latency

In contrast to lytic infection, during latency the viral genome is assembled into
nucleosomes (27) and is maintained as a circular episome (117, 148, 149). The promoter
and the enhancer of the LAT gene associate with higher levels of H3 K9/K14ac relative to

the transcriptionally inactive ICPO gene in mouse models of latency (92, 93). Similarly,

21

another active transcription mark, H3K4me2, was enriched on the LAT enhancer when
compared with IE gene promoters in latently infected rabbit neurons (46). Conversely,
during the establishment of latency, viral lytic genes progressively associate with

H3 K9me, indicative of heterochromatin formation on the viral genome (185). Prevention
of heterochromatin spreading into the LAT region is thought to be mediated in part by
CTCF and the insulator—like elements upstream of LAT promoter and in the LAT intron
(3). In the course of reactivation from latency by explantation of infected mouse dorsal
root ganglia, concomitant with the decrease in LAT RNA abundance, H3 K9/K14ac
association decreases on the LAT enhancer but increases on the now transcriptionally
active ICPO promoter (2). Consistent with these findings is the observation that inhibition
of HDAC activity by intraperitoneal sodium butyrate injection also results in acetylation
of histones on the lytic genes and reactivation from latency in ocularly-infected mice

(128).

Several groups have attempted to recapitulate in vivo latent infections by
employing cell culture-based quiescent infections established either by infection of
fibroblasts by replication-defective HSV-1 (S8) or by differentiating rat
pheochromocytoma cells (PC 12) into neurons and infecting with wild-type HSV-1 in the
presence of acyclovir (22). Using the former model, the HSV-1 genome was found to
associate with heterochromatin protein HP], but not other heterochromatin marks such as
H3K9me (36). On the other hand, another study using a Similar model showed that
quiescent viral genomes associated with high levels of H3K9me3, and upon reactivation
from quiescence, increasing levels of acetylated histone H3 were present on lytic

promoters (19). In the second model of quiescence, HDAC inhibitors stimulated the

22

production of infectious virions, suggesting a role for histone acetylation in reactivation
of viral gene expression (23). In contrast, trichostatin A did not increase de-repression of
quiescent HSV-1 genomes in human fibroblasts (130, 141, 173). These observations
collectively indicate that in most systems histone modifications and the transcriptional
activity of the viral genome correlate with each other. What remains uncertain is whether
histone modifications cause changes in viral gene expression or if, vice versa, changes in
gene expression result in altered histone patterns. In either case, the mechanistic details

remain to be uncovered.

Factors that mediate the changes in chromatin on the viral genome during latency

and reactivation from laterfl

Abundant evidence has established that, during HSV-1 latency, lytic gene
expression is repressed and the viral genome (with the exception of the LAT gene)
associates with heterochromatin. However, the mechanisms that mediate these changes in
the chromatin structure of the viral genome during establishment of and reactivation from
latency remain poorly defined. The components of the VP16-induced complex (VIC) and

LAT are leading candidates as potential mediators of these processes.

Given that IE gene transcription is repressed in latently infected neurons, various
studies have addressed whether latency is a result of inhibiting VIC formation on IE gene
promoters. Repression of IE gene expression during latency cannot be solely attributed to
the absence of VPl 6, as ectopic expression of VP16 did not prevent the establishment of
latency in mice infected from the ocular route (160). This same study found that ectopic

expression of VP16 was not sufficient to induce reactivation from latency as indicated by

23

the absence of infectious viruses in tears and in the explanted trigeminal ganglia on
infected mice (160). In contrast, another group reported that, when expressed from
adenoviral vectors, VP16, ICPO, and ICP4 can each induce reactivation from latency in
explanted trigeminal ganglia (56). Given that VP16 is phosphorylated on multiple serines
(134), differential phosphorylation of VP16 in neuronal cells may lead to repression of IE

gene expression during latency, although no evidence currently supports this possibility.

Other studies have indicated that the inhibition of IE gene expression may be due
to the modulation of VIC components other than VP16, namely HCF-l and Oct-l. HCF-l
is localized to the cytoplasm in sensory neurons in vivo, but is transported to the nucleus
under conditions that induce reactivation of latent HSV-1 (89). Interestingly, a recent
study indicated that HCF-l localizes to Golgi apparatus in unstimulated sensory neurons,
and disruption of Golgi by brefeldin A treatment leads to accumulation of HCF -1 in the
nucleus, indicating that regulation of HCF-l localization may be an important factor
between the transition from latent to lytic infection (82). Given that HCF-l was shown to
be responsible for nuclear import of VP16 (96), it will be interesting to address whether
VP16 is sequestered in the cytoplasm upon infection of neuronal cells and whether VP16
is translocated to the nucleus upon reactivation from latent infection. Another mechanism
that may explain the repression of IE gene transcription is the low level of Oct-l
expression in ganglionic sensory neurons (55, 60). Although one hypothesis suggests that
competition of Oct-2 with Oct-1 for binding to IE promoters might repress IE gene
expression (74, 102), very low levels of Oct-2 expression in sensory neurons argue

against this possibility (SS, 60).

24

As explained in detail above, during latency, the viral genome takes a form
resembling heterochromatin with the exception of the actively transcribed LAT gene.
Interestingly, absence of LAT expression correlates with an increase in euchromatin and
decrease in heterochromatin marks on the viral genome (185), suggesting that LAT
expression may be required for the heterochromatin formation on the viral genome. In
addition, absence of LAT resulted in an increase in lytic gene expression during latency
(16, 41, 114). Therefore, it is important to distinguish whether LAT represses lytic gene
expression by directly inducing the heterochromatin formation or indirectly by inhibiting
lytic gene transcription, which may also lead to heterochromatinization of the viral
genome. Since LAT accumulates in the nucleus at high levels without being localized to
distinct foci that contain the viral genomes, it seems unlikely that LAT is directly
involved in heterochromatin formation on the HSV-1 genome. The recent identification
of LAT-encoded miRNAs that target IE genes (122, 179) may explain how LAT induces
the repression of lytic gene expression and formation of heterochromatin on the viral
genome. Although LAT may participate in regulation of chromatin on the latent viral
genome, it is important to note that not all latently infected neurons express LAT (1 16,
157) and that absence of LAT does not preclude establishment of latency (69, 99).
Therefore, LAT itself cannot not be the only factor that regulates viral chromatin during

latent infection.

1.2.3. Chromatin during the infection of other herpesviruses

The preceding sections detail the current state of understanding of the regulation
of chromatin during both lytic and latent stages of HSV-1 infection. Other herpesviruses

are also subject to silencing by heterochromatin formation on the viral genome during

25

latent infections and overcome this chromatin barrier during reactivation from latency or
lytic infections. The following paragraphs will summarize some of these mechanisms in
human cytomegalovirus (HCMV or HHV-S), Kaposi’s sarcoma-associated herpesvirus

(KSHV or HHV-8), and Epstein-Barr virus (EBV or HHV-4) infections.

Cell culture (66) and ex vivo (145) model systems of HCMV infection, as well as
in vivo murine CMV (MCMV) infections (106), have all indicated a mode of chromatin
regulation similar to that of HSV-1 in many respects. For instance, upon establishment of
latency, the major IE promoters of MCMV and HCMV associate with HPl (106, 123,
145), HDACS (106, 123), and histones that carry inactive transcription marks (66, 106,
145). In contrast, during productive infection and reactivation from latency, the major IE
promoter is associated with acetylated histones (66, 106, 123, 145). Other findings also
support the idea that histone acetylation might play an important role in HCMV
infections. First of all, HDAC inhibitors increase the permissiveness of otherwise
nonpermissive cells for viral infection (123). Second, during lytic infection, virion protein
pp71 induces the degradation of Daxx, a component of ND10 domains that repress
transcription through HDACS (152, 153). Third, the major IE proteins IE72 and IE86
interact with and block the activity of HDACS (129). Finally, the IE86 protein interacts
with p300/CBP (64) and PCAF HATS (10) in order to modulate the cell cycle and
potentiate the transcription of viral genes, respectively. These findings are consistent with
a common theme observed in HSV-1 infections: during lytic infection, various viral
factors block the formation of heterochromatin on the viral genome and recruit
transcriptional coactivators that covalently modify the histones and induce active

transcription.

26

The Rta/ORFSO protein of KSHV is the main viral transcriptional activator
protein and triggers the latent-to-lytic infection switch in KSHV-infected cells. Like
VP16 of HSV-1 , Rta/ORFSO interacts with CBP, which augments the transcriptional
activity of Rta in a heterologous expression system (54). Although our knowledge about
chromatin and histone modifications on the KSHV genome during lytic and latent
infections is limited, a few studies indicated that histone acetylation may play a role. For
instance, reactivation of KSHV from latency can be mediated by sodium butyrate, an
HDAC inhibitor, leading to dissociation of latency-associated nuclear antigen (LANA)
from the ORFSO promoter (1 12, 113). Concomitant with the dissociation of LANA, the
ORFSO promoter associates with acetylated histones and the Brg-l chromatin remodeling
complex (112, 113). LANA mediates transcriptional repression and heterochromatin
formation on the viral genome likely by interacting with the mSin3A co-repressor
complex (91), HP] (104), and SUV39H1 histone methyltransferase (155). Although
LANA and LAT are not genetically homologous, the repressor function of LANA
resembles that of LAT, which induces heterochromatin formation on the HSV-1 genome
by a currently elusive mechanism (185). A distinct feature of LANA is its interaction
with histone H2A-H2B dimers, which mediates the maintenance of KSHV episomes
during latency (4). An intriguing possibility is that this interaction between LANA and
histone H2A-H2B may also contribute to transcriptional regulation by LANA.
Interestingly, LANA also interacts with CBP; however, this interaction results in the
inhibition of the HAT activity of CBP (103). The model emerging from these findings is
that KSHV chromatin is regulated dynamically during lytic and latent stages of infection

in a way similar to that of HSV-1. Yet while many studies have focused on histone

27

acetylation and deacetylation as the major switch between lytic and latent KSHV
infections, other covalently modified histones that correlate with active transcription,
such as H3K4me3, might also associate with the viral genome. Future work should more
completely define the KSHV chromatin and identify the factors that mediate these

changes.

A number of viral antigens that are expressed during EBV latency interact with
some of the same transcriptional coactivators that VP16 associates with. For instance,
EBV nuclear protein 2 (EBNA2), an essential protein for latency and B-cell
immortalization, associates with the p300, CBP, and PCAF HATS (184), as well as with
components of the Brg-l chromatin remodeling complex (188), which all contribute to
EBNA2’s transactivation potential. Interestingly, EBNA3C, another critical component
for EBV-mediated B-lymphocyte immortalization, can act as both an activator and a
repressor of transcription depending on its interaction partners at a given promoter. For
instance, EBNA3C interacts with both transcriptional coactivators such as p300 (166) and
co-repressors including HDACS (79, 143) and mSin3A (79). However, it is currently not
known whether recruitment of these coactivators by EBNA2 and EBNA3C results in

covalent modification of histones on target promoters.

Other EBV proteins, such as BRLF l (Rta) and BZLFl (Zta), which induce the
switch from latent to lytic infection, also interact with CBP for enhanced transcription (1,
167, 197). Recruitment of CBP by Zta to IE promoters results in increased histone
acetylation (26), suggesting an active role for histone modifications in mediating
reactivation from latency. Others have observed similar correlations between the

transcriptional status of EBV genes and histone modifications, in particular histone

28

acetylation, during different stages of infection. For instance, during latency, the
promoter of latent membrane protein 2A (LMP2A) is enriched in acetylated histone H3
and H4 and in H3K4me2, the levels of which correlate with the amount of LMP2A
transcript (43). In contrast, the promoter of the transcriptionally inactive BZLFl gene is
silenced by recruitment of class II HDACS during latency (50). The transcriptional
activity of the LMPl and EBNA2 promoters during latency also correlates with presence
of active or inactive histone marks (14, 24). The hypothesis that histone acetylation
contributes to the switch from latent to the lytic cycle is supported by the observation that
the HDAC inhibitor TSA resulted in an increase in the levels of acetylated histone H4 on
the Rta promoter and induced expression of the viral lytic proteins Rta and Zta (13).
However, a more detailed study indicated that although HDAC inhibitors can increase the
levels of histone acetylation on viral lytic gene promoters, they were not sufficient to
trigger reactivation from latency (21). Therefore, regulation of the switch fiom latent to
lytic infection may not be simply explained by histone modifications, in spite of the

striking correlation between transcriptional activity and the state of chromatin.

These findings clearly indicate that in all herpesviruses, during lytic infection,
actively transcribed viral genes are either devoid of histones or associate with histones
that carry active transcription marks. In contrast, during latency, most viral genes are not
transcribed and are packaged in a form resembling heterochromatin. Strikingly, most of
the changes during the switch between latent and lytic infections by various herpesviruses
are associated by the recruitment of similar host factors, such as p300 and C BP HATS. In

return, the host cell tries to block herpesvirus infections by silencing the viral genome

29

mainly by recruitment of HDACS and HPl to the viral DNA. Whether other mechanisms

are involved in this tug of war will be the subject of future research.

30

2. PERSPECTIVES

The prevailing evidence at this time clearly establishes that various herpesviruses
target similar components of the host’s transcriptional machinery, despite differences in
the composition of their genomes. Another emerging theme is the correlation between the
transcriptional status of the viral genomes and the histone marks that associate with those
genomes. However, these correlations should not necessarily be interpreted as
representing causal relationships. In other words, histone modifications may not be the
cause of the switch from latent to lytic herpes infection, but rather a result of
transcriptional activity. Moreover, given that histone modifications are mediated by
enzymes that are recruited by specific DNA binding proteins such as transcriptional
activators, it is of crucial importance to identify the cellular or viral factors that bring
about these changes in the state of viral chromatin, rather than relying on histone

modifications as being the sole determining factors.

Despite the often implicit or tacit assumptions, viral gene expression during lytic
infection may in fact not be regulated by mechanisms Similar to those that govern cellular
genes, perhaps highlighted by the fact that histones are not deposited at high levels on the
viral genome to begin with. In contrast, during reactivation from latency, the viral
genomes are heavily nucleosomal and as such resemble cellular genes. Therefore,
reactivation from latency is more likely to be mediated by mechanisms similar to those

that activate cellular genes.

A potential limitation in the analysis of viral chromatin is the ChIP technique and

how the data obtained in these assays should be interpreted. For instance, during lytic

31

infection one would expect that not all viral genomes will enter the host cell nuclei at the
same time and not all of them will be activated transcriptionally. Yet, what precipitates in
the IP reaction will be a population of these heterogenous viral genomes, which although
not testable, are assumed to immunoprecipitate at similar efficiencies. The problem of
heterogeneity becomes even more problematic during reactivation from latency, where
only a small fraction of viral genomes may reactivate and as such it may be difficult to
assay the changes in the chromatin structure of this small fraction of reactivating viral
genomes. Therefore, care should be taken while interpreting the results obtained from

ChIP assays of infected cells.

Another concept that warrants further investigation is the mechanism(s) by which
histones are depleted from the viral genome during lytic infection. Although some studies
indicated that active transcription marks are present on the viral genome during lytic
infection, the density of histones on viral DNA seems far lower than on cellular genes.
Likely candidates in this process include the histone chaperones and assembly factors.
For example, the histone chaperone HIRA is localized to PML bodies in senescent cells
(194). A recent study has indicated that HIRA might be involved in the deposition of
H33 on the HSV-l genome, and disruption of HIRA expression impaired viral gene
expression and replication modestly (137). According to this study, histone deposition by
HIRA might be necessary for optimal viral gene expression during lytic infection.
Considering that histones are under-represented on the HSV-1 genome, future studies are
necessary to address whether HIRA or other histone chaperones are important for viral
gene expression or whether they are direct targets of HSV-1 proteins during lytic

infection.

32

Although histone deposition by chaperones and removal by chromatin-remodeling
enzymes is one potential mechanism to account for the low density of histones on viral
DNA, an alternative is that histones may not be deposited at all on a large fraction of the
viral genomes, and thus histone modifications may not matter for viral gene expression.
In line with this idea, disrupting the expression of various transcriptional coactivators that
are recruited by VP16 had no substantial effect on viral IE gene expression (95). To date,
little is known about how histone deposition on viral genomes is prevented during lytic

infection.

Another potential mechanism of histone depletion from the HSV-1 genome
during lytic infection is transcription by RNAP II itself. The rate of transcription by
RNAP II correlates with depletion of histones (61, 94). Consistent with this model are
recent observations that inhibition of RNAP II transcription leads to a gradual increase in
histone occupancy on the HSV-l genome (SK, unpublished observations). This would
also be consistent with the notion that histone changes on viral DNA might be a

consequence, rather than a cause, of changes in viral gene expression.

Although we have a better picture of the regulation of chromatin on the HSV—l
genome during lytic and latent stages of infection, we are far from understanding how the
changes on HSV-1 chromatin are mediated and whether they matter for different stages
of infection. Therefore, future studies are necessary to explore whether alternative

mechanisms explained above operate during herpes infections.

33

3. ACKNOWLEDGEMENTS

We apologize to colleagues whose original work was not cited because of space
limitations. This work was supported in part by NIH grant AI-064634 to SJ T, a
predoctoral fellowship from the Greater Midwest Affiliate of the American Heart
Association to SBK, and the Van Andel Research Institute. Thanks to Dr. Xu Lu and

David Nadziejka for their comments on the manuscript.

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52

CHAPTER TWO

Kutluay, Sebla, B., DeVos, Sarah, L., Klomp, Jennifer, E., Triezenberg, Steven, J. (2009).
Transcriptional Coactivators are not Required for Herpes Simplex Virus Type 1
Immediate Early Gene Expression in vitro. Journ_al of Virology, 83(8): 343 6-49.

53

Chapter 2

ROLE OF TRANSCRIPTIONAL COACTIVATORS IN HSV-l
INFECTION

1. ABSTRACT

Virion protein 16 (V P16) of herpes simplex virus type 1 (HSV-1) is a potent
transcriptional activator of viral immediate early (IE) genes. The VP16 activation
domain can recruit various transcriptional coactivators to target gene promoters.
However, the role of transcriptional coactivators in HSV-1 IE gene expression during
lytic infection had not been fully defined. We have shown previously that transcriptional
coactivators such as the p300 and CBP histone acetyltransferases and the BRM and Brg-l
chromatin remodeling complexes are recruited to viral IE gene promoters in a manner
mostly dependent on the presence of the activation domain of VP16. In this study, we
tested the hypothesis that these transcriptional coactivators are required for viral IE gene
expression during infection of cultured cells. Disrupted expression of the histone
acetyltransferases p300, CBP, PCAF, or GCNS or the BRM and Brg-l chromatin
remodeling complexes did not diminish IE gene expression. F urthennore, IE gene
expression was not impaired in cell lines that lack functional p300, or BRM and Brg-l .
We also tested whether these coactivators are required for VP 1 6-dependent induction of
IE gene expression from transcriptionally inactive viral genomes associated with high
levels of histones in cultured cells. We found that disruption of coactivators also did not
affect IE gene expression in this context. Thus we conclude that the transcriptional

coactivators that can be recruited by VP16 do not contribute significantly to IE gene

54

expression during lytic infection or induction of IE gene expression from nucleosomal

templates in vitro.

55

2. INTRODUCTION

Herpes simplex virus type 1 (HSV-1) is a large double-stranded DNA virus that
establishes life-long latency in sensory neurons after an initial phase of lytic infection in
epithelial cells. Viral gene expression during lytic infection is initiated by VP16, a
tegurnent-associated transcriptional activator protein that stimulates the transcription of
viral immediate early (IE) genes (6). VP16 is recruited to viral IE gene promoters through
cis-regulatory elements with a consensus sequence of 5’ TAATGARAT, in association
with two host‘cell proteins, Oct-1 and HCF -1 (74). VP16, by its activation domain (AD),
interacts with various general transcription factors and recruits the host RNA polymerase
II (RNAP II) (12, 24, 29, 43, 75).

The packaging of eukaryotic DNA in the form of chromatin presents a significant
impediment to the transcriptional machinery (42). This barrier can be overcome by
activator-dependent recruitment of coactivator protein complexes with either of two types
of enzymatic activities. Some coactivators covalently modify histones by acetylation,
methylation, phosphorylation, ubiquitinylation, sumoylation, ADP-ribosylation or proline
isomerization (30, 42). Some covalent histone modifications, such as acetylation of lysine
9 and lysine 14 of histone H3 (H3K9/K14ac) or trimethylation of lysine 4 of histone H3
(H3K4me3), are marks of active transcription. In contrast, methylation of other lysine
residues on histones is typically indicative of inactive transcription and heterochromatin
formation (30). The second class of coactivators hydrolyzes ATP in the process of
remodeling the position of nucleosomes along DNA or in removing nucleosomes from
DNA (11, 59).

The interaction of transcriptional activators with coactivators has often been

56

explored using a chimeric protein, Gal4-VP16 (5 8), comprising the DNA-binding domain
of the Saccharomyces cerevisiae Gal4 protein and the activation domain (AD) of HSV-1
VP16. The VP16 AD can physically interact with and recruit transcriptional coactivators
such as the histone acetyltransferases (HATS) p300 (KAT3B) and CBP (KAT3A) (3, 17,
25, 34, 67, 70), PCAF (KAT2B) (70) and GCNS (KAT2A) (23, 38, 63, 67, 68), or the
ATP-dependent chromatin remodeling enzymes BRM and Brg-l (16, 46, 49, 50) to
potentiate transcription from nucleosomal templates. However, the role of coactivators in
the context of HSV-1 infection is not yet well-defined, in part because of prior evidence
that the HSV-l genome is predominantly non-nucleosomal during lytic infection (40, 41,
47).

We and others have recently shown that histones, most often represented
experimentally by histone H3, are present on the HSV-1 genome during lytic infection,
but at lower levels than cellular genes (20, 22, 28, 35, 52). Furthermore, active
transcription marks such as H3K9/K14ac and H3 K4me3 have been associated with viral
genes during lytic infection (20, 22, 28, 35). We have also shown that, at early times
during lytic infection, the p300 and CBP HATS and the BRM and Brg-l chromatin
remodeling enzymes are recruited to viral IE gene promoters in a manner mostly
dependent on the presence of VP16 AD (20). Similarly, the Set] histonc
methyltransferase, which is recruited by HCF-l, was shown to contribute to optimal
HSV-1 gene expression (22). These results suggest that, during lytic infection,
nucleosomes might be deposited on the viral genome, and yet recruitment of
transcriptional coactivators could result in modification and removal of the histones from

the viral genome similar to actively transcribed genes in the host cell genome (4, 19, 33,

57

56).

Based on this model, we have hypothesized that the transcriptional coactivators
that are recruited by VP16 are required for IE gene expression during lytic infection.
From this hypothesis, we predicted that disrupting the expression of a coactivator will
diminish IE gene expression by allowing formation of an inactive chromatin structure on
the viral genome. Here we show that, contrary to our hypothesis, disrupting the
expression of various coactivators by RNA interference (RNAi) did not decrease IE gene
expression in HSV-infected cells under most conditions tested. In parallel with these
findings, IE gene expression was not impaired in SiHa cells, which do not express
functional p300, or in SW13 and C33-A cells neither of which express the BRM and Brg-
l remodeling enzymes. Moreover, restoration of BRM and Brg-l activity to SW13 or
C33-A cells had no substantial effect on IE gene expression, indicating that neither BRM
nor Brg-l remodeling enzymes are essential for IE gene expression.

If not important for lytic infection, we then hypothesized that coactivators may be
required during reactivation from latency, during which the viral genomes are
nucleosomal. We have not yet tested the requirement of coactivators during reactivation
from latency in vivo; instead, we used in vitro conditions in which viral genomes are
heavily occupied with nucleosomes in cultured cells. To this end, we employed a mutant
virus strain (RPS) that lacks sequences encoding the activation domain of VP16. During
RPS lytic infection, IE gene expression is reduced dramatically (66, 76) and histones
associate with the RPS genomes at higher levels than the wild-type genomes (20).
Moreover, p300 and CBP HATS or BRM and Brg-l remodeling enzymes are not

recruited efficiently to RPS IE promoters (20). Although IE gene expression from the

58

RPS genome was induced significantly upon superinfection by HSV-2, none of the
transcriptional coactivators were required for this induction. We conclude that the
coactivators that are recruited by VP16 are not essential for IE gene expression during

lytic infection in vitro, regardless of the nucleosomal status of the viral genome.

59

3. METHODS
Cell lines and viruses: HeLa (ATCC# CCL-2), SW-13 (ATCC# CCL-105), Vero
(ATCC# CCL-81) and telomerase-transformed human foreskin fibroblasts (HF F 3)
provided by Wade Bresnahan, were grown in Dulbecco’s modified Eagle medium
(Invitrogen) containing 110 mg/l sodium pyruvate and 10 % fetal bovine serum
(Invitrogen). SiHa (ATCC# HTB-3 5) and C33-A (ATCC# HTB-31) cells were grown in
minimum essential medium containing Earle’s salts supplemented with non-essential
anrino acids, 1 mM sodium pyruvate and 10 % fetal bovine serum. The RPS strain of
HSV-1, which lacks sequences encoding the activation domain of VP16, and the RPSR
(RPS rescue) strain have been previously described (66). HSV-1 strains KOS, RPSR and
RPS and HSV-2 strain G were prepared and titers were determined using Vero cells. In
some experiments, cycloheximide (100 pg/ml) was added to the medium for 30 minutes

prior to and during infection to inhibit protein translation.

Plasmids and transfections: A p300 expression plasmid, pCI-FLAG-p300, was
provided by Yoshihiro Nakatani (5). The pCG-BRM, pBJ-Brg-l, dnBRM and dnBrg-l
expression plasmids were obtained from Bernard Weissman and David Reisman (1, 64).
SiHa cells were transfected using jetPEl (Polyplus) transfection reagent according to the
manufacturer’s instructions. SW13 and C33-A cells were transfected with Lipofectarnine

2000 (Invitrogen) according to the manufacturer’s instructions.

siRNAs and transfections: For each target coactivator, two siRNA duplexes were

purchased from Qiagen with the exception of CBP_1 (Dharmacon). The catalogue

60

numbers and sequences of siRNAs are given in Table 1. For siRN A transfections,
1.5x1035 HHFs were plated per well in 6-well cell culture plates one day prior to
transfection. SiRN A duplexes were transfected at 10 nM (for single and double
transfections) or 20 nM (for quadruple transfections) total concentration using Silentfect
transfection reagent (Bio-Rad) according to manufacturer’s instructions, with the
exception that the siRNA duplexes and the transfection reagent were diluted in

OPTIMEM reduced-serum medium (Invitrogen).

Gene expression assays and Q-RT-PCRs: Total cellular RNA was isolated using Trizol
reagent (Invitrogen). Total RNA was reverse-transcribed using random primers in a
commercial reverse transcription system (Promega). The cDNA was used as template in
quantitative real time PCR (Q-RT-PCR) assays using SYBR Green Master Mix (Roche)
and ABI 7500 Real-Time PCR System (Applied Biosystems). Gene expression was first
normalized against 18S rRNA and then to appropriate controls by the 2'AACt method. For
chromatin immunoprecipitation assays data was analyzed using the standard curve
method explained in more detail in the following section. Primer pairs used in this study
are indicated in Table 2. Other primer pairs have been previously described (20, 54). For
statistical analysis of gene expression, four or more biological replicates of a given

experiment were analyzed by Student’s t-test.

Immunoblotting: Total cell lysates were prepared by RIPA buffer (50 mM Tris, pH 7.4,
150 mM NaCl, lmM EDTA, 1 % Triton X—100, l % sodium deoxycholate, 0.1% SDS)

supplemented with protease inhibitors (10 mM PMSF, 5 ug/ml aprotinin and leupeptin).

6]

25-50 pg of the lysates were run on 6, 8 or 10% SDS-PAGE and transferred to PVDF
membranes. Blots were blocked in 5 % nonfat dry milk-TTBS (100 mM Tris pH 7.5, 150
mM NaCl, 0.1% Tween 20) overnight at 4 °C under constant agitation. The blots were
then incubated for two hours with the primary antibodies diluted in TTBS supplemented
with l % BSA (Invitrogen). Antibodies specific for p300 (SC-S84), CBP (SC-369), BRM
(SC-6450), Brg—l (SC-10768), PCAF (SC-13124) and GCNS (SC-6303) were obtained
from Santa Cruz Biotechnology. Antibodies specific for CD44 (156-3C11) and GAPDH
(ab9484) were purchased from Cell Signaling Technology and Abcam, respectively.
Blots were then incubated with the proper HRP-conjugated secondary antibodies and
visualized using a chemiluminescence detection system (Pierce) and Chemi-Doc Imaging

System with Quantity One software (Bio-Rad).

Chromatin immunoprecipitation: Chromatin immunoprecipitation was performed as
explained before with minor modifications (20). Briefly, confluent plates of HFF cells
were infected with RPS or RPSR strains of HSV-1 at an MOI of 0.025 pfu/cell and S
pfu/cell, which corresponded to about 8-10 viral genomes per cell for each infection. At 6
hpi infections were stopped by addition of formaldehyde to cell culture plates at a final
concentration of 1 %. Chromatin was isolated and sonicated using Branson Digital
Sonifier-450 to obtain 200-1000 bp DNA fragments. Protein-DNA complexes were
immunoprecipitated using 5 pg of antibodies against histone H3 (Abcam, ab1791).
Protein-DNA complexes were collected by Protein G-agarose beads (Invitrogen). After
several washes, the protein-DNA complexes were eluted and reverse-crosslinked

overnight at 65°C, in the presence of 200 mM NaCl and 10 ug RNaseA. Samples were

62

then precipitated with ethanol, digested with proteinase K (Roche) at 42 °C for 2 hours
and purified with Qiagen spin columns using the gel extraction protocol. The presence of
viral and cellular DNA fragments in the immunoprecipitated material was analyzed by
quantitative real-time PCR using SYBR Green Master Mix (Roche) and ABI 7500 Real-
Time PCR system (Applied Biosystems). A standard curve using serial 3-fold dilutions of
input samples (1, 0.3, 0.1, 0.04 %) was produced to quantitate the Signals from
immunoprecipitation samples. Background signals, obtained from immunoprecipitation
reactions performed in the absence of antibodies (no antibody control), were subtracted
from signals obtained from immunoprecipitation samples [referred to as “% input (IP-
noab)]. When necessary, data was further normalized against the cellular control U3
snRN A promoter, by dividing the “% input (IP-noab)” value for the viral DNA by that of

the cellular DNA, to account for the differences in immunoprecipitation efficiencies.

63

4. RESULTS

RNAi of p300 and CBP HA Ts does not diminish HS V-I IE gene expression:

The related HAT enzymes p300 and CBP can potentiate VP16 AD-dependent
transcriptional activation from reconstituted nucleosome arrays in vitro or from reporter
plasmids in vivo (3, 17, 67, 70). We have previously shown that both p300 and CBP are
recruited to IE gene promoters in a manner mostly dependent on the presence of VP 1 6
AD (20). Others have also indicated that p300 and CBP are recruited to PML bodies that
become viral replication compartments later in infection (37, 44, 45). However, a direct
role for p300/CBP in viral gene expression has not yet been established. The present
study was undertaken to address this gap in our understanding.

We have previously shown that disrupting the expression of p3 00 by plasmid-
based RNAi in HeLa cells did not affect IE gene expression (35). However, this finding
was complicated by several considerations. First, p300 and CBP in some contexts have
been shown to be redundant (26, 69) and therefore knocking down p300 itself may not
have been sufficient to affect IE gene expression. Second, RNAi is not 100 % efficient,
and thus the residual levels of p300 might have been sufficient for IE gene expression.
Third, analysis of coactivators in HeLa cells might be inherently flawed due to the
presence in HeLa cells of the papillomaviral proteins E6 and E7, which affect the
activities of p300 and CBP (55, 79).

To overcome these potential problems, we disrupted the expression of p300 and
CBP, both separately and in combination, by multiple siRNA duplexes in telomerase-
transfonned human foreskin fibroblasts (HF F s). Steady-state levels of p300 and CBP

protein (Fig. 2.1A) and mRNA (Fig. 2. 1 B) were significantly and specifically reduced by

siRNA duplexes designed to target these two related proteins. One siRN A duplex
targeting p300 (p300_1) reduced p300 protein expression more than p300 mRN A
expression, which likely reflects a block in mRN A translation rather than mRN A
degradation.

We then tested whether IE gene expression was reduced by disruption of p3 00
and CBP in HFFS. To address this, HF FS were transfected with siRNA duplexes as
described above and were then infected with wild-type HSV-1 at a multiplicity of
infection (MOI) of 10 plaque-forming units per cell (pfu/cell). At 2.25 hours post
infection (hpi), when IE gene expression is robust, total RNA was isolated and further
processed for analysis of IE gene expression. To our surprise, disruption of neither p300
nor CBP resulted in reduced expression of the ICPO, ICP4 or ICP27 mRNAs (Fig. 2. 1 C).
Moreover, even simultaneous knockdown of p300 and CBP had no deleterious effect on
viral IE gene expression. One of the p300-specific siRNA duplexes (p300_2), either
alone or in combination with the CBP_2 duplex resulted in a statistically Significant
increase in IE gene expression, quite the contrary of the expected outcome. We suspect
that this increase represents an off-target effect of that specific siRNA, as the other
duplex that also targets p300 (p300_1) did not Show a similar effect.

The initial assays shown in Fig. 2.1C were conducted using relatively high M01
(10 pfu/cell). We considered whether a requirement for p300 and CBP might be more
evident during low multiplicity infections, in which the viral genome might be more
prone to transcriptional repression by deposition of host histones. HFF cells transfected
with the various SiRNA duplexes were infected at low M01 (0.] pfu/cell) and RNA

harvested at 4 hpi was analyzed for IE gene expression using quantitative RT-PCR (Fig.

65

2.1D). The results (Fig. 2.1D) are comparable to the high MOI infections; siRNAs
targeting p300 or CBP (or both together) have no deleterious effect on viral IE gene
expression. In fact, ICPO and ICP27 but not ICP4 expression showed a modest but
statistically significant increase when p300 and CBP are knocked down either separately
or together. These results suggest that p300 and CBP are not required for IE gene
expression at low M01, and if anything, they may act to repress IE gene transcription.
The IE gene products ICP4 and ICPO themselves have activities that regulate IE
gene transcription; for example, ICPO might bypass the requirement for coactivators by
disrupting the REST/CoREST/HDAC repressor complex (13, 14). To prevent any
feedback on IE gene expression by the IE proteins themselves, parallel experiments were
conducted in the presence of cycloheximide, a translation inhibitor. The results (Fig.
2.1E) again Show that siRNAs targeting p300 or CBP have no deleterious effect on viral
IE gene expression. Disruption of p300 by the p300_2 duplex, either alone or in
combination with CBP_2, caused a statistically significant increase in expression of all IE
genes during low multiplicity infections (Fig. 2. 1 D, B), an observation that contradicts
our original hypothesis. This increase is likely due to an off-target effect of the p300_2
siRNA duplex, since the other p300 siRNA did not have a similar effect. We conclude
that the HATS p300 and CBP are neither required nor redundant for activation of IE gene

expression by VP16.

66

Figure 2.1: Disruption of p300 and CBP expression by RNAi does not decrease HSV-1
IE gene expression. Human foreskin fibroblasts (HFFS) were transfected with siRNA
duplexes targeting p300, CBP or a negative control non-targeting siRNA duplex. (A)
Immunoblot Showing p3 00, CBP and GAPDH protein levels 48 hours after siRNA
transfection. Arrow indicates the CBP-specific band. (B) Q-RT-PCR analysis of p300
and CBP expression in siRNA-transfected and KOS-infected HFFS. Values for each
target are represented relative to the negative control siRNA signal. Histograms represent
the average of 6 independent experiments and error bars represent the standard deviation.
(C) siRNA-transfected HFFS were infected with HSV-1 KOS strain at an MOI of 10
pfu/cell. Expression of viral IE genes (ICPO, ICP4, ICP27) at 2.25 hours post infection
was analyzed by Q-RT-PCR. (D) siRNA-transfected HFFS were infected with HSV-1 at
an MOI of 0.1 pfu/cell. IE gene expression at 4 hpi was analyzed by Q-RT-PCR. (E)
siRNA-transfected HFFS were pretreated with 100 ug/ml cycloheximide for 30 minutes
and then infected with HSV-1 at an MOI of 0.1 pfu/cell in the presence of 100 ug/ml
cycloheximide for 4 hours. IE gene expression was analyzed by Q-RT-PCR. Data in
panels C, D and E represent the average of two independent experiments, each done with
biological duplicates. Error bars represent the standard deviation based on these four
samples. Mean values that differ significantly from those obtained from cells transfected
with negative control siRNA are indicated by C“) for p<0.01 or by (#) for 0.01<p<0.05 as
determined by Student’s t test.

67

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68

IE gene expression is not impaired in SiHa cells but is augmented by expression
of wild-type p300:

Although p300 was knocked down efficiently by siRNAs in HF FS, we were
concerned that the residual expression of p300 might still be sufficient to enable IE gene
expression. To address this, we analyzed IE gene expression in SiHa cervical carcinoma
cells, which express a mutated form of p300 that lacks the bromodomain (53). Since both
SiHa and HeLa cells are derived from cervical carcinomas and are transformed by human
papillomaviruses, we reasoned that comparing IE gene expression in these two cell lines
would be a legitimate approach to test whether p3 00 is required for transcription of IE
genes. SiHa and HeLa cells were infected with KOS at l pfu/cell and IE gene expression
at 2 hours post-infection was analyzed by Q-RT-PCR. Contrary to our hypothesis, IE
gene expression in SiHa cells was not significantly different than HeLa cells (Fig. 2.2A).
We also tested whether supplementing SiHa cells with fully functional p3 00 might
further enhance viral IE gene expression. To that end, SiHa cells were transfected with a
wild-type p300 expression plasmid or an empty plasmid. Overexpression of wild-type
p300 in SiHa cells (Fig. 2.2B) resulted in increases in ICPO, ICP4 and ICP27 expression
that were modest but statistically significant (Fig. 2.2C), suggesting that although p300 is

not required, it may potentiate the transcription of viral IE genes in SiHa cells.

69

A HIGH I ICP27 B

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33 0.4

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E 1.0
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Figure 2.2: HSV-1 IE gene expression is not impaired in SiHa cells but is augmented in
the presence of wild-type p300. (A) HeLa and SiHa cells were infected with HSV-1
strain KOS at an MOI of 1 pfu/cell. IE gene (ICPO, ICP4, ICP27) and p300 mRNA levels
at 2 hpi were analyzed by Q-RT-PCR. Values for each gene tested in SiHa cells are
represented relative to HeLa cells. Error bars show the range between the averages of two
independent experiments. (B) SiI-Ia cells were transfected with 2.5 ug of pCI (empty) or
pCI-p300 plasmids. p300 and GAPDH expression was analyzed 24 hours after
transfection by immunoblotting. (C) SiHa cells were transfected as in (B) and were
infected with HSV-1 KOS strain at an MOI of 5 pfu/cell. IE gene expression (ICPO,
ICP4, ICP27) at 2 hpi was analyzed by Q-RT-PCR. Values for each viral gene tested in
pCI—p3OO transfected cells are represented relative to cells transfected with empty
plasmid (pCI). Error bars indicate standard deviations (n = 4). Mean values that vary
significantly (p< 0.01 , Student’s t test) from those obtained from cells transfected with

vector plasmid are indicated (*).

70

Disruption of PCAF and GCN5 HA Ts does not affect HS V-I IE gene
expression:

In addition to p300 and CBP, other HATS such as PCAF and GCN5 are known to
interact with the activation domain of VP16 (68, 70). We therefore considered whether
PCAF and GCN5 might be preferentially required for IE gene expression. To address
this, IE gene expression was analyzed in HFFS in which the expression of PCAF and
GCN5 was diminished either separately or together by siRN A duplexes. The siRNAs
targeting PCAF and GCN5 in HF F s were both effective and specific for their respective
targets, as indicated by immunoblotting (Fig. 2.3A) and Q-RT-PCR (Fig. 2.3B). We then
infected these cells with HSV-l at high M01 (10 pfu/cell) and analyzed [B gene
expression at 2.25 hpi. As we observed for p300 and CBP, IE gene expression was not
affected significantly under most circumstances when PCAF and GCN5 (separately or in
combination) were knocked down (Fig. 2.3C). Transfection of two siRNAs (PCAF_1 and
GCN5_2) caused a slight but statistically significant decrease in IE gene expression.
However, since comparable effects were not exhibited by the PCAF__2 and GCN5_1
duplexes, which block target protein expression to similar levels as do the PCAF_1 and
GCN5_2 duplexes, we cannot attribute these modest changes in IE gene expression to the
effects on disruption of PCAF or GCN5. Parallel experiments were performed at low
MOI (0.1 pfu/cell), to see whether a requirement for PCAF and GCN5 might be more
apparent under conditions of a lighter viral genome load. In most cases, IE gene
expression did not change significantly when PCAF and GCN5 expression was reduced
(Fig. 2.3D). Although the GCN5_1 duplex reduced IE gene expression, GCN5_2 duplex

caused the opposite effect. Therefore, although these siRNAs alter IE gene expression,

71

we do not think that these modest effects on IE gene expression are biologically
meaningful. To rule out the possibility that IE proteins themselves were masking a
requirement for coactivators in the activity of VP16 on IE gene expression, we repeated
the low MOI experiments in the presence of cycloheximide to block IE protein
expression. Although the decrease in ICPO and ICP4 expression when GCN5 was
knocked down (with either of two siRNA duplexes) was statistically significant, in
general the expression of IE genes was not impaired when PCAF and GCN5 were
knocked down either separately or in combination (Fig. 2.3E). Collectively, these results
suggest that although VP16 can recruit PCAF and GCN5 in artificial contexts, PCAF and
GCN5 are neither required substantially nor redundant for IE gene transcription during
lytic infection.

To address whether different classes of HATS are redundant for IE gene
expression, we then simultaneously disrupted the expression of p300, CBP, PCAF and
GCN5 HATS in HF Fs, and infected these cells at high (10 pr/CCll) or low (0.1 pfu/cell)
MOI with HSV-1. As indicated in Figure 2.3F, regardless of the MOI, disruption of all
four HATS did not reduce viral IE gene expression; if anything, we observed a slight but
significant increase in ICPO and ICP27 expression. These results suggest that p300, CBP,
PCAF and GCN5 HATS are not redundant for IE gene expression during lytic infection in

vitro.

72

Figure 2.3: RNAi of PCAF and GCN5 does not decrease HSV-1 IE gene expression.
HFFS were transfected with siRNA duplexes targeting PCAF, GCN5 or a negative
control non-targeting siRNA duplex. (A) Western blots Showing levels of PCAF, GCN5
and GAPDH proteins 48 hours after siRNA transfection. (B) Q-RT-PCR analysis of
PCAF and GCN5 expression in siRNA-transfected and KOS-infected HFFS. Data
represent the average of six independent experiments, and error bars represent the
standard deviation. (C) siRNA—transfected HF Fs were infected with HSV-1 at an MOI of
10 pfu/cell. Total RNA was isolated at 2.25 hpi and IE gene mRNA levels (ICPO, ICP4,
ICP27) were analyzed by Q-RT-PCR. (D) siRNA-transfected HFFS were infected with
HSV-1 at an MOI of 0.1 pfu/cell. IE gene expression at 4 hpi was analyzed by Q-RT-
PCR. (E) siRNA-transfected HFFS were pretreated with 100 ug/ml cycloheximide for 30
minutes and then infected with HSV-l at an MOI of 0.1 pfu/cell in the presence of
cycloheximide. IE gene expression at 4 hpi was analyzed by Q-RT-PCR. Panels C-E
present the average of four replicates, and error bars represent standard deviations. (F)
HFFS were transfected with siRNA duplexes targeting p300, CBP, PCAF, GCN5 or a
negative control non-targeting siRNA duplex and infected with HSV-1 at an MOI of 10
or 0.1 pfu/cell. IE gene expression at 2.25 and 4 hpi for high and low MOI, respectively,
were analyzed by Q-RT-PCR. Data shown are from a representative experiment done
with biological triplicates; error bars indicate the standard deviation. Experimental
samples whose mean values differ significantly from those obtained from cells
transfected with negative control siRNA are indicated by (*) for p<0.01 or by (#) for
0.01<p<0.05 as determined by Student’s t test.

73

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RNAi of BRM and Brg-I chromatin remodeling complexes does not affect IE
gene expression:

Several lines of evidence indicate that ATP-dependent chromatin remodeling
complexes can be recruited by the activation domain of VP16 to nucleosomal templates
leading to disruption of nucleosomes and transcriptional activation (46, 49, 50, 72, 78).
VP16 AD can also enhance nucleosome eviction by the SWI/SNF remodeling complex
from mononucleosomal templates in vitro (16). Therefore, we hypothesized that BRM
and Brg-l chromatin remodeling complexes, the mammalian homologues of the yeast
SWI/SNF complex (72), might remove the nucleosomes fiom the viral genome and
enable active transcription.

To address this, as in previous sections, we analyzed IE gene expression in HFFS
in which the expression of BRM and Brg-l remodeling enzymes was disrupted by RNAi,
with the expectation that IE gene expression would diminish in the absence of BRM and
Brg-l . Immunoblotting (Fig. 2.4A) and Q-RT-PCR (Fig. 2.4B) results indicate that BRM
and Brg-l were knocked down very efficiently and Specifically by both siRNA duplexes
against each target. However, disruption of BRM and Brg-l expression by most siRN A
duplexes (with the exception of Brm_1) did not reduce IE gene expression at high M01
(10 pfu/cell) during lytic infection (Fig. 2.4C). To the contrary, IE gene expression was
increased in the presence of some siRNAs, most notably Brg-l_2. In parallel
experiments performed at low MOI (0.1 pfu/cell), transcription of some IE genes during
lytic infection was affected by one but not by both of the siRNA duplexes targeting either
BRM or Brg-l (Fig. 2.4D). Therefore, we conclude that reduced levels of the BRM or

Brg-l remodeling enzymes do not affect IE gene transcription substantially. Moreover,

75

similar results were also obtained when the viral infection was performed in the presence
of cycloheximide to prevent the interference of IE proteins in IE gene transcription (Fig.
2.4B). Interestingly, under all conditions tested, expression of most IE genes increased
Significantly when Brm and Brg-l were knocked down together (Fig. 2.4C, D, E),
suggesting that Brm and Brg-l might be acting redundantly in a manner contrary to our
original hypothesis, i.e., to inhibit rather than to support IE gene expression. Although
primarily associated with transcriptional activation, Brm and Brg-l have been Shown to

potentiate transcriptional repression by the Rb tumor suppressor protein (15).

76

Figure 2.4: Disruption of BRM and Brg-l expression does not decrease HSV-l IE gene
expression. HFFS were transfected with the indicated SiRN A duplexes targeting BRM,
Brg-l or a negative control non-targeting siRNA duplex. (A) Western blot Showing
BRM, Brg-l and GAPDH expression in HF Fs 48 hours after siRNA transfection. (B) Q-
RT-PCR analysis of BRM and Brg-l in HF Fs after siRNA transfection and KOS
infection. Data represent the average of six independent experiments. Error bars represent
the standard deviation. (C) After siRNA transfection, HFFS were infected with HSV-1 at
an MOI of 10 pfu/cell. IE gene expression at 2.25 hpi was analyzed by Q-RT-PCR. (D)
siRNA-transfected HF Fs were infected with HSV-1 at an MOI of 0.1 pr/CCII. IE gene
expression at 4 hpi was analyzed by Q-RT-PCR. (E) After siRNA transfection, HFFS
were pretreated with 100 ug/ml cycloheximide for 30 minutes and then infected with
HSV-1 at an MOI of 0.1 pfu/cell in the presence of 100 ug/ml cycloheximide. Total RNA
was isolated at 4 hpi and IE gene expression was analyzed by Q-RT—PCR. C to E present
the mean of four biological replicates; error bars represent standard deviations. Mean
values that differ significantly from those obtained from cells transfected with negative
control siRNA are indicated by (*) for p<0.01 or by (#) for 0.01<p<0.05 as determined
by Student’s t test.

77

of .> V 'E';
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Brg-1 ~__. 7 _ 3:: 3’
GAPDH - 6.3“ 9t“ f’avafas’“
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DICPO IICP4 IICP27

    

relative exproulon

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o\ > a» > 9 ~
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78

IE gene expression in cell lines with defective BRM and Brg-I expression:

As a complementary strategy to RNAi, we also analyzed IE gene expression in
cell lines that do not express BRM and Brg-l enzymes (57, 73). The immunoblot shown
in Figure 2.5A confirms that the levels of BRM and Brg-l proteins are dramatically
reduced in SW13 (adrenal carcinoma) and C33-A (cervical carcinoma) cells as compared
with HeLa cells.

We then asked whether IE gene expression was impaired in SW13 and C33-A
cells. Parallel cultures of HeLa, SW13 and C33-A cells were infected with HSV-1 at high
(5 pfu/cell) or low (0.1 pfu/cell) MOIS. Q-RT-PCR assays of viral IE gene expression
revealed no defect in SW13 cells and about a 2-fold increase in C33-A cells in the high
multiplicity infections (Fig. 2.5B). At low MOI (0.1 pfu/cell), IE gene expression was
about 60 % lower in SW13 cells, but 2-fold higher in C33-A cells (Fig. 2.5C). Given that
both cell lines are defective for BRM and Brg-l , we cannot attribute the deficit in SW13
cells at low MOI to a requirement for these coactivators.

To further test for potential contributions by BRM and Brg-l , we transfected
SW13 or C33-A cells with plasmids expressing either wild—type BRM or Brg-l or
dominant-negative forms that lack ATPase activity (Fig. 2.5D, G). The endogenous gene
encoding the cell surface marker CD44 served as a positive control, since CD44 is known
to be regulated by BRM and Brg-l in these cells (64, 65). As expected, CD44 mRNA
expression was induced in both SW13 and C33-A cells upon expression of BRM and
Brg—l but not the dominant negative BRM and Brg-l (Fig. 2.5E, H), indicating that the
BRM and Brg-l proteins ectopically expressed in these cell lines are functional. When

these cells were subsequently infected with HSV-1, IE gene expression in SW13 cells

79

that express wild-type BRM and Brg-l was not significantly different (p> 0.05, by
Student’s t-test) than cells that were transfected with an empty plasmid or with plasmids
encoding the dominant negative forms of BRM or Brg—l (Fig. 2.5F). Curiously, IE gene
expression in C33-A cells expressing wild-type BRM or Brg-l was approximately 60%
lower than in parallel cells transfected with empty vector (Fig. 2.51). However, this
reduction seems independent of the catalytic activity of BRM and Brg—l, Since expression
of the dominant-negative forms of BRM and Brg-l also reduced IE gene expression to
similar levels (Fig. 2.51). This suggests that BRM and Brg-l do not repress IE gene
expression, as suggested above by RNAi assays (Fig. 2.4). In other words, if BRM and
Brg-l were inhibitory for IE gene expression, restoring BRM and Brg-l expression in
both SW13 and C33-A cells would decrease IE gene expression. These results together
with the RNAi assays described above lead us to conclude that the chromatin remodeling
enzymes BRM and Brg-l are neither required nor redundant for IE gene expression

during lytic infection.

80

Figure 2.5: HSV-l IE gene expression in SW13 and C33-A cells that do not express
BRM and Brg-l remodeling enzymes. (A) BRM, Brg-l and GAPDH expression in HeLa,
SW13 and C33-A cells was analyzed by immunoblotting. (B, C) HeLa, SW13 and C33-A
cells were infected in parallel at an MOI of 5 pfu/cell (B) or 0.1 pfu/cell (C). IE gene
expression (ICPO, ICP4, ICP27) at 2 hpi was analyzed by Q-RT-PCR. IE gene expression
in SW13 and C33-A cells is represented with respect to that in Hela cells. The graph
shows the average of two independent experiments each done in biological quadruplicate
(B) or triplicate (C). Error bars represent the range between the averages of these
experiments. Mean values that vary significantly (p< 0.01, Student’s t test) from those
obtained from HeLa cells in both of the experiments presented in (B) and (C) are
indicated (*). (D) SW13 cells were transfected with 4 ug of an empty plasmid or with
expression plasmids encoding BRM, Brg-l, dominant-negative BRM (dnBRM) or
dominant negative Brg-l (dnBrg-l) together with 0.5 pg of puromycin selection plasmid.
24 hours post-transfection, media was replaced by puromycin selection media (2.5 ug/ml
puromycin). After 2 days of puromycin selection, total protein was isolated and analyzed
by immunoblotting against BRM, Brg-l and GAPDH. (E, F) SW13 cells were
transfected with the indicated plasmids as in (D) and infected with HSV-l KOS at an
MOI of 0.1 pfu/cell. At 2 hpi total RNA was isolated and, CD44 (E) or IE gene
expression (F) was analyzed by Q-RT-PCR. (G) Immunoblot showing BRM, Brg-l,
CD44 and GAPDH expression in C33-A cells transfected with the indicated plasmids as
in (D). (H, I) C33-A cells were transfected with the indicated plasmids as in (D) and
infected with HSV-1 KOS at an MOI of 0.1 pfu/cell. At 2 hpi total RNA was isolated and
CD44 (panel H) and IE gene expression (panel I) were analyzed by Q-RT-PCR. Panels E,
F, H, and I are derived from a representative experiment done with biological triplicates;
error bars represent standard deviation. Mean values that vary significantly (p< 0.01,
Student’s t test) from those obtained from cells transfected with vector plasmid are
indicated (*).

81

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Coactivators are not required for VP16-mediated induction of IE gene
expression from nucleosomal viral genomes in vitro:

One potential reason why transcriptional coactivators are not required for IE gene
transcription during lytic infection is that the viral genomes remain depleted of histones
by some undefined mechanism that may bypass the need for coactivators. To explore this
question, we designed an experiment in which histones can be more abundantly deposited
on viral genomes prior to the introduction of transcriptionally-active VP16. We have
shown previously that IE gene expression is dramatically reduced during lytic infection
by RPS, a mutant virus that lacks sequences encoding the activation domain of VP16 (66,
76). In addition, recruitment of the p300 and CBP HATS or the BRM and Brg-l
chromatin remodeling enzymes to most IE promoters is also impaired in RPS infections
(20). Furthermore, histones (represented by histone H3) associate with RPS genomes to a
greater extent than with wild-type genomes (20). Therefore, to some extent RPS infection
resembles quiescent or latent infections with respect to defects in IE gene expression and
increased histone occupancy on the viral genome.

We first asked whether IE genes in the RPS genome could be activated by
superinfection with HSV-2, which encodes a VP16 protein very similar to that of HSV-1
(9) and which can induce reactivation from quiescence in other contexts (8). HFFS were
infected with RPS at MOIS ranging from 0.0005 pfu/cell to 0.05 pfu/cell, which
correspond to approximately 0.5-1 viral genome per cell to 50-100 viral genomes per
cell, respectively (data not shown). At 6 hours post-infection, histone deposition on RPS
genomes was dramatically higher than on wild-type genomes (Fig. 2.6A). The RPS-

infected cells were then superinfected with HSV-2 strain G at MOIS ranging from 0.1

83

pfu/cell to 10 pfu/cell. Two hours after initiating the HSV-2 infection, we assayed levels
of HSV-l-specific IE gene expression by Q-RT-PCR. As expected, superinfection of
RPS- infected cells with HSV-2 activated the expression of ICP4 (Fig. 2.6B) and ICP27
(Fig. 2.6C) in a dose-dependent manner with respect to HSV-2 MOI. These results
indicate that the defect in IE gene expression in RPS infections can be overcome
effectively by providing VP16 in trans by HSV-2 superinfection. In subsequent assays
we have performed RPS and HSV-2 infections at MOIS of 0.005 pfu/cell and 10 pfu/cell,

respectively, to obtain robust IE transcription.

84

A B C
Histone H3 ChIP ICP4 ICP27
+ RPS: 0.00 + RPS: 0.05
-- RPS: 0.005 -- RPS: 0.005
1-6 a RP"; -o- RPS: 0.000S + RPS: 0.000S
1 2 IRP5 5 100 10000
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3 i :m
- M .5 ’ g 10
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gflf‘tfié *9 a?" ' 0.1 1 10 0.1 1 1o
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Figure 2.6: HSV-2 superinfection induces IE gene expression in RPS-infected cells. (A)
HFFS were infected with RPS or RPSR strains of HSV-1 at an MOI of 0.025 pfu/cell or 5
pfu/cell, respectively. At 6 hpi ChIP was performed assaying the presence of histone H3
on the ICPO promoter, ICP27 promoter, ICP27 ORF, tk promoter and gC promoter. (B,C)
HFFS were infected with RPS strain of HSV-1 at an MOI of 0.05, 0.005 or 0.0005
pfu/cell. At 6 hpi of RPS infections, superinfection with HSV-2 was performed at MOIS
of 0.1, 1 or 10 pfu/cell. ICP4 (B) and ICP27 (C) expression at 2 hpi of HSV-2
superinfection was analyzed by Q-RT-PCR.

85

To address whether coactivators are required for HSV-2-mediated expression of
IE genes from the quiescent RPS genomes, we disrupted the expression of each
coactivator in HFFS by siRNAs, infected these cells with RPS and then superinfected
with HSV-2. Contrary to our hypothesis, IE gene expression from the RPS genomes was
effectively stimulated by HSV-2 superinfection, even in the presence of siRNAs targeting
any of the coactivators (Fig. 2.7). If anything, the siRNAs resulted in a modest increase,
rather than a decrease, in IE gene expression. These results suggest that coactivators are
not required for VP16-mediated induction of IE gene transcription from viral DNA that is

abundantly associated with histones.

86

Figure 2.7: Coactivators are not required for HSV-2-dependent induction of IE gene
expression from RPS genomes. HFFS were transfected with the indicated siRNA
duplexes targeting (A) p300 and CBP, (B) PCAF and GCN5, (C) BRM and Brg-l or a
negative control non-targeting siRNA duplex. 48 hours after transfection, HFFS were
infected with RPS at an MOI of 0.005 pfu/cell. At 6 hpi of RPS infection, HF Fs were
superinfected with HSV-2 at an MOI of 10 pfu/cell. HSV-1 IE gene expression at 2 hpi
of HSV-2 infection was analyzed by Q-RT-PCR. The graph displays the mean of four
biological replicates, and error bars denote standard deviations. Mean values that differ
significantly from those obtained fiom cells transfected with negative control siRNA are
indicated by C“) for p<0.01 or by (#) for 0.01<p<0.05 as determined by Student’s t test.

87

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88

5. DISCUSSION

Although a number of prior reports described evidence that the HSV-l genome
remains non-nucleosomal during lytic infection (40, 41, 47), several groups have recently
shown that the viral genome is not exclusively histone-free and that acetylated and
methylated histones are present on the viral genome during lytic infection, albeit at lower
levels than on cellular genes (20, 22, 28, 35, 52). In contrast, during latency, the viral
genome is packaged in a manner more nearly resembling host cell chromatin (10).
Histones on the latent HSV-l genomes carry post-translational modifications typical of
heterochromatin, with the exception of the actively-transcribed latency-associated
transcript gene (32, 71). During reactivation from latency or quiescence, the histones
associated with IE gene promoters and other regions of the genome become acetylated, a
hallmark of transcriptionally active chromatin (8, 51). These observations all indicate that
regulation of chromatin might play a crucial and distinct role in different stages of HSV-1
infection.

We do not yet understand how the viral genome manages to stay predominantly
histone-free during lytic infection. Given that VP16 AD interacts with a number of
transcriptional coactivators in artificial conditions and that some of these coactivators are
recruited to IE gene promoters during lytic infection (20), we hypothesized that these
coactivators are involved in establishing a transcriptionally active chromatin state on the
viral genome and enable IE gene expression. To this end, we tested whether disrupting
the expression of these coactivators would decrease IE gene expression. Reducing the
expression levels of p300 and CBP HATS, singly or in combination, using siRNAs had

no discernible effect on IE gene expression at different multiplicities of infection (Fig.

89

2.1), suggesting that neither p300 nor CBP are required for IE gene expression. The
modest increase in IE gene expression observed following transfection of one of the p300
duplexes, p300_2, is attributed to an off-target effect of this siRNA duplex, since the
other siRNA was just as effective in diminishing p300 protein levels but had no effect on
viral IE mRNA levels. An alternative explanation that we cannot exclude is that the
diminished expression of p300 seen in the presence of siRNA p300_2 might suppress the
host innate immune defense and hence enhance IE gene expression as suggested by
others (45). Disruption of other HATS, PCAF and GCN5, by RNAi also had no
significant effect on IE gene expression under most conditions tested in vitro (Fig. 2.3).
Together, these results lead us to reject the hypothesis that histone acetylation is required
for IE gene expression during lytic infection in vitro in HFFS.

Given that the activation domain of VP16 can stimulate nucleosome eviction by
the yeast SWI/SNF remodeling complex (16), we hypothesized that BRM and Brg-l , the
mammalian homologues of SWI/SNF, may be required for removing the nucleosomes
from the viral genome. However, disruption of BRM and Brg-l , separately or together,
did not reduce IE gene expression (Fig.2. 4). Although RNAi of BRM and Brg-l together
increased the expression of most IE genes in some conditions, these results were not
supported by our findings in SW13 and C33-A cells. IE gene expression was not
impaired in SW13 and C33-A cells, which express neither BRM nor Brg-l (Fig. 2.58, C)
and restoring BRM and Brg-l expression in these cell lines did not increase IE gene
expression (Fig. 2.5F, I). Collectively, these results indicate that BRM and Brg-l do not
have a substantial role in supporting the VP16-dependent activation of viral IE gene

expression during lytic infection in vitro.

90

The results of these experiments suggest that the transcriptional coactivators
tested here are not required for IE gene expression during lytic infection, in contrast with
our initial hypothesis. We recognize that cell culture models of HSV-1 infection are not
necessarily representative of in viva infections in epithelial or neuronal cells. Therefore,
future studies will be necessary to test whether coactivators are important in lytic
infection in vivo.

One potential explanation for the lack of an effect is redundancy or compensation
among coactivators, such that the activity lost by disruption of one coactivator is taken up
by another coactivator. We have addressed this in part by analyzing IE gene expression in
HFFS where two or more coactivators were simultaneously disrupted. However, even
when as many as four coactivators were targeted by siRNAs, viral IE gene did not
decrease (Fig. 2.3F). Nonetheless, we cannot fully exclude the possibility that other
HATS or remodeling complexes are compensating for those disrupted in our experiments.

We used immunoblots of the targeted proteins as an indication of the effectiveness
of the siRNAs employed in these experiments. Although the targeted protein levels were
substantially diminished (to levels 20% or less relative to control cells), the residual
protein may be sufficient for the biological activities we have tested. Unfortunately, in
most cases, no suitable positive control genes have been identified; that is, genes that are
known to be direct targets of a given coactivator in HFFS. The exception may be the
CD44 gene in SW13 and C33-A cells, which clearly responded to the presence of wild-
type but not mutant forms of Brg-l or BRM. In addition, as suggested in a recent study
(21), we observed that disruption of p300 and CBP HATS together led to a substantial

decrease in H3Kl8ac (Figure 2.8), suggesting that our RNAi of CBP and

91

p300 expression was effective. To gain confidence in the outcomes of the siRNA
experiments, we also took the complementary approach of testing IE gene expression in
mutant cell lines that lack the particular coactivator. The consistency of the results from
these two approaches strongly supports our conclusion that these transcriptional

coactivators are not intimately involved in HSV-1 IE gene expression.

92

H3K1 8ac DAPI H3K14ac DAPI

Figure 2.8: H3K18 but not H3K14 acetylation is reduced when p300 and CBP expression
is disrupted together. HF F s were transfected with siRNA duplexes targeting p300 and
CBP or with a negative contral siRN A duplex as in Figure 2.1. At 2 days post-
transfection, immunofluorescence assays were performed as explained before (21).

(-)control

 

8i p300+CBP

93

Although the coactivators tested in this study are not required for IE gene
expression, other coactivators might be required for modification of chromatin structure
on the viral genome. Employing similar assays, others have shown that the Setl histone
methyltransferase can be recruited by HCF -1 and can contribute to viral gene expression
during lytic infection (22). Although disruption of Setl expression did not significantly
affect IE gene expression at early times in infection, at later stages viral gene expression
and viral replication were reduced modestly. Similarly, during lytic infection by varicella
zoster virus (like HSV-1, a member of the alphaherpesvirus family), Setl recruitment to
. the IE62 promoter was correlated with high levels of H3 K4me3 (48). However, whether
Setl is required for IE62 expression has not been explicitly tested. Therefore, further
studies are necessary to ask whether Setl or other coactivators are important for IE gene
transcription.

A further possibility is that VP16 recruits transcriptional coactivators not for IE
gene expression during lytic infection, but during reactivation from latency where the
viral genome is nucleosomal (10). We have addressed this inpart by testing whether
coactivators are required during activation of IE gene expression from histone-laden viral
genomes in vitro. HSV-1 strain RPS lacks the VP16 activation domain and the density of
histone H3 on the RPS genome approaches that of cellular genes at later times in
infection (Fig. 2.6A). Superinfection by HSV-2 of RPS-infected cells resulted in
substantial IE expression from the RPS template (Fig. 2.6). However, knocking down
expression of various coactivators had little or no effect on RPS IE gene expression (Fig.
2.7), suggesting that VP16-mediated reactivation of IE gene expression does not require

the coactivators tested in this study. We recognize that this in vitro system is not a

94

genuine representation of latent infection and that some experiments from others have
suggested that VP16 may not be required for reactivation (62). Nonetheless, these
experiments do address the role of coactivators for VP16-mediated transcription from a
predominantly nucleosomal template. Future studies will be necessary to more directly
establish whether coactivators are required in other quiescent infection models or, most
importantly, during reactivation from latency in vivo.

A final possibility is that these coactivators may not be required at all for HSV-1
gene expression; their apparent presence at IE gene promoters may simply reflect their
association with larger complexes of the transcription machinery. Recruitment of a
particular transcription factor does not always correspond to a functional requirement for
that factor. For instance, although estrogen receptor-a can bind to a large number of
cellular promoters, only about 10% of those genes are actually regulated by estrogen
(36).

Given that the transcriptional coactivators tested in this study are not required for
IE gene transcription, the mechanism by which VP16 stimulates IE gene transcription is
still not clear. Although current models suggest that histones are first deposited on the
viral genome at early times in infection and then removed, no evidence indicates that
histone deposition precedes IE gene transcription. An alternative to the model that
histones are deposited, modified and then removed is a model in which deposition of
histones on the viral genome is prevented by a yet undefined mechanism. If this is the
case, then coactivators would not be required for transcription of IE genes. Alternatively,
other mechanisms, such as recruitment of Set] histone methyltransferase by HCF -1 (22,

48) or disruption of REST/CoREST/HDAC repressor complex by ICPO (l3, 14), may be

95

the major determinant for histone depletion on the viral genome. In support of ICPO
having a role in chromatin dynamics during HSV-1 infections, a recent study indicated
that the absence of ICPO resulted in an increase in histone occupancy and a decrease in
the ratio of acetylated histones on the viral genome during lytic infection (7). Since we
have minimized ICPO protein expression in our assays, it is unlikely that ICPO will have
bypassed the need for VP16 —dependent recruitment of coactivators in the present work.
An alternative mechanism that might lead to histone depletion is high rates of
transcription by RNAP II. Several studies in yeast have indicated that histones are
depleted fi'om heavily transcribed regions (19, 31, 33, 39, 61). Certain histone
chaperones, such as Spt6 and FACT, are components of the RNAP II transcription
machinery and facilitate elongation by RN AP 11 (2, 27). By extension then, VP16, ICPO
and ICP4 might all contribute to effective transcription by RNAP II, which may in turn
result in histone depletion from the viral genome. Another histone chaperone potentially
involved in histone dynamics on the viral genome is HIRA, which is implicated in
replication-independent histone deposition (18, 60) and was Shown to be present in PML
bodies in senescent cells (77). Future studies Should test whether RNAP II or the
associated histone chaperones underlie histone depletion from the HSV-l genome.
Overall, we have shown in this report that various transcriptional coactivators are
not required for IE gene expression during lytic infection or during VP16-mediated
induction of IE gene transcription from nucleosome-laden HSV-1 genomes in cultured
cells. Future studies should address whether other transcriptional coactivators contribute
to viral gene transcription during lytic infection. The underlying mechanism for histone

deposition on or histone removal from the HSV-1 genome must still be defined. Finally,

96

the role of chromatin dynamics during establishment and reactivation of latent infections

remains an important and incompletely answered question.

6. ACKNOWLEDGEMENTS
BRM, Brg-l, dnBRM and dnBRG-l expression plasmids were generously provided by
Bernard Weissman and David Reisman. pCI-p300 plasmid was obtained from Yoshihiro
Nakatani. We thank Rick Hay and Wade Bresnahan for providing SW13 and telomerase-
transforrned human foreskin fibroblasts, respectively. We thank Dr. Carrie Graveel, Dr.
Xu Lu, and Glen Alberts for comments on the manuscript. This research was supported
by the Department of Biochemistry and Molecular Biology at Michigan State University,
the Van Andel Research Institute and NIH grant AIO64634. SK was supported by a
special fellowship from the College of Natural Science at Michigan State University and
by a predoctoral fellowship from the American Heart Association. SD was supported by a

summer research internship funded by the Fred and Lena Meijer Foundation.

97

 

 

10.

11.

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104

Table 1. siRNA sequences and catalogue numbers used in this study.

 

siRNA

Catalog #

siRNA sequence

 

p3 00_1

p3 00_2

CBP_l

CBP_2

BRM_1

BRM_2

BRG4_1

BRG-1_2

PCAF_1

PCAF_2

GCN5_1

GCN5_2

AllStars
control

1024846

8102626267

Custom

designed“

8102622648

8100726747

8103033191

8100047579

8103098998

8103038035

8103048325

81004261 18

81004261 25

1027280

(8) CCC CUC CUC UUC AGC ACC AdeT*
(A) UGG UGC UGA AGA GGA GGG GdeT

(8) GGA CUA CCC UAU CAA GUA AdeT
(A) UUA CUU GAU AGG GUA GUC CdAdA

(S) AAC UGU CGG AGC UUC UAC GAG deT
(A) CUC GUA GAA GCU CCG ACA GUU deT

(S) CGC AUUGUC GAA CCA UGA AdeT
(A) UUC AUG GUU CGA CAA UGC GdeG

(S) GCG UCU ACA UAA GGU GUU AdeT
(A) UAA CAC CUU AUG UAG ACG CdCdT

(S) GCC CAU CGA UGG UAU ACA UdeT
(A) AUG UAU ACC AUC GAU GGG CdeT

(S) GCG CUA CAA CCA GAU GAA AdeT
(A) UUU CAU CUG GUU GUA GCG CdeG

(8) GGG CGU ACG AGU UUG ACA AdeT
(A) UUG UCA AAC UCG UAC GCC CdAdG

(S) AGU CUA CCU CGG UAC GAA AdeT
(A) UUU CGU ACC GAG GUA GAC UdeT

(8) CGC CGU GAA GAA AGC GCA AdeT
(A) UUG CGC UUU CUU CAC GGC GdAdT

(S) CCA UUU GAG AAA CCU AAU AdeT
(A) UAU UAG GUU UCU CAA AUG GdAdG

(8) GCG GCA UCA UCG AGU UCC AdeT
(A) UGG AAC UCG AUG AUG CCG CdeG

Sequence information not provided.

* dN represents the 3’ deoxyribonucleic acid overhangs.
** Synthesized by Dharmacon.

105

 

Table 2. Oligonucleotides used as primers in quantitative real-time PCR . Other PCR

primers used in this study are as explained before (20, S4).

 

 

Gene Oligonucleotide sequence
p300 (F) 5’-CAATGAGATCCAAGGGGAGA
(R) S’-ATGCATCTTTCTTCCGCACT
CBP (F) 5’-GTGCTGGCTGAGACCCTAAC
(R) S’-GGCTGTCCAAATGGACTTGT
BRM (F) S’-CTGAAGATCGTGCTGCTTTG
(R) 5’-CCAGTCGCTGTCAAAGATGA
BRG-l (F) S’-TCACTGACGGAGAAGCAGTG
(R) S’-TTCTTGCTCTCGTCGTCCTI‘
PCAF (F) S’-GAAACTGGAGAAACTCGGAGTGTAC
(R) 5’-TI‘TCCAGCCATTACA'ITTACAAGACT
GCN5 (F) 5’-TCCTCACTCACTTCCCCAAATT
(R) 5’-TGGAGAGTTTGCCCCATAGATC
CD44 (F) 5’-GAAACTGGAACCCAGAAGCACA
(R) 5’-TGATGCTCATGGTGAATGAGGG
ICP27 (F) 5’-TGGTGTCTGATTGGTCCTTG
promoter (R) S’-CGGGTGGTGGATGTCCTTAT
gC (F) S’-TCGGGCGATTGATATA‘1”I 1 1'1

promoter

(R) 5’-TGTCCCCTTCCGGAATTTAT

 

106

CHAPTER THREE

Kutluay, Sebla B., Doroghazi, James, Roemer, Martha, Triezenberg, Steven J.
(2008). Curcumin Inhibits Herpes Simplex Virus Immediate Early Gene Expression by a
Mechanism Independent of p300/CBP Histone Acetyltransferase Activity. Virology.
373(2): 239-47.

107

Chapter 3

CURCUMIN INHIBITS HSV-l INFECTION BY A MECHANISM
INDEPENDENT OF P300/CBP HAT ACTIVITY

1. ABSTRACT

Curcumin, a phenolic compound from the curry spice turmeric, exhibits a wide
range of activities in eukaryotic cells, including antiviral effects that are at present
incompletely characterized. Curcumin is known to inhibit the histone acetyltransferase
activity of the transcriptional coactivator proteins p300 and CBP, which are recruited to
the immediate early (IE) gene promoters of herpes simplex virus type 1 (HSV-1) by the
viral transactivator protein VP16. We tested the hypothesis that curcumin, by inhibiting
these coactivators, would block viral infection and gene expression. In cell culture
assays, curcumin significantly decreased HSV-l infectivity and IE gene expression.
Entry of viral DNA to the host cell nucleus and binding of VP16 to IE gene promoters
was not affected by curcumin, but recruitment of RNA polymerase II to those promoters
was significantly diminished. However, these effects were observed using lower
curcumin concentrations than those required to substantially inhibit global H3
acetylation. No changes were observed in histone H3 occupancy or acetylation at viral
1E gene promoters. Furthermore, p300 and CBP recruitment to IE gene promoters was
not affected by the presence of curcumin. Finally, disruption of p300 expression using a
short hairpin RNA did not affect viral IE gene expression. These results suggest that
curcumin affects VP16-mediated recruitment of RNA polymerase II to [B gene promoters

by a mechanism independent of p300/CBP histone acetyltransferase activity.

108

 

2. INTRODUCTION

Curcumin (diferuloylmethane) is the major component of the curry Spice turmeric
(Curcuma longa Linn.). Curcumin can affect the metabolism of cells and organisms in a
number of ways, including apoptosis, cell signaling, inflammation and carcinogenesis
[reviewed in (22, 45)]. Various antiviral effects of curcumin have been described, but the
biochemical mechanisms of those effects have been incompletely defined (6, 7, 31 , 36,
47). Curcumin reportedly inhibits transcription and replication of the human
immunodeficiency virus (HIV-1) by blocking Tat-mediated transactivation or by
diminishing viral protease and integrase activity (7, 31, 36, 47). Virions of herpes
simplex virus type 2 (HSV-2) were rendered less infectious by exposure to curcumin
prior to infection of HeLa cells or of mouse genitalia (6). In some cases, the antiviral
effects of curcumin arise from inhibition of a cellular process or a transcription factor.
For example, curcumin has been shown to suppress transcription activation by the host
protein AP-l (2) leading to diminished HTLV-I and HPV-mediated cellular
transformation (10, 42, 48). i

The packaging of eukaryotic DNA in the form of chromatin presents a significant
impediment to the transcriptional machinery (30). This barrier can be overcome by the
action of coactivator proteins that typically comprise multi-protein complexes with either
of two types of enzymatic activities. Some coactivators covalently modify histones by
acetylation, methylation, phosphorylation, ubiquitinylation, prolyl isomerization or ADP-
ribosylation (17, 21, 33, 37, 40, 51). Other coactivators hydrolyze ATP in the process of
remodeling the position of nucleosomes along DNA or in removing nucleosomes from

DNA (23, 46). Curcumin inhibits the histone acetyltransferase (HAT) activity of the

109

closely-related transcriptional coactivator proteins p300 and CBP in vitro (ICso of 25 uM)
and in vivo (3, 34). This inhibition might disrupt expression of genes dependent on those
coactivators.

Herpes simplex virus type 1 (HSV-1) is a large DNA virus with a linear double
stranded genome of 152 kb. Upon entry by fusion of its envelope with the host cell
plasma membrane, the viral capsid is transported to the nuclear pore and the viral genome
is released into the nucleus which starts the temporally regulated gene expression cascade
involving transcription of immediate-early (IE or a), early (E or B) and late (L or 7)
genes. IE gene transcription is strongly stimulated by the virion-bome trans-activator
VP16 (8, 53). VP16 comprises a core domain (residues 1-410) and an acidic activation
domain (AD) (residues 413—490), which has often been used for creating novel
transactivators in heterologous expression systems (44, 49). VP16 is recruited to
TAATGARAT motifs on IE gene promoters through interactions of its core domain with
two host proteins, HCF and Oct-1, forming the VP16-induced complex (VIC) (55).
VP16, mainly by its activation domain, then interacts with various general transcription
factors and recruits RNA polymerase II (RNAP II) machinery (13, 18, 25, 32, 52, 56).
Although the HSV-1 genome has long been suggested to be non-nucleosomal during lytic
infection (28, 29, 38, 39), we and others have recently reported that histone H3 is
associated with the HSV-1 genome and that histone tail modifications such as
methylation and acetylation can be detected on transcriptionally active viral promoters
and ORFS (15, 16, 24). We have also shown that p300 and CBP HATS are recruited to IE
gene promoters in a manner dependent on the VP16 AD (15), suggesting that histone

acetylation by p300/CBP on the viral genome might be important for [B gene expression.

110

 

 

Since curcumin inhibits p300/CBP HAT activity, we wanted to test whether
curcumin could act as a potential anti-herpetic compound by inhibiting IE gene
expression. In this report, we show that curcumin treatment of HeLa cells at non-toxic
levels slows HSV-1 replication and decreases the ability of cells to support infection by
HSV-1. This effect is mediated at least in part by inhibition of IE gene expression, since
curcumin reduced the recruitment of RN AP 11 to IE gene promoters but not to Cellular
control promoters. However, the concentrations of curcumin required for these effects on
viral gene expression were far less than the concentrations required to substantially
inhibit global H3 acetylation. Moreover, the occupancy of histone H3 on IE gene
promoters and the acetylation of H3 at those promoters did not change Significantly in the
presence of curcumin. Furthermore, no effect on viral gene expression was observed
when p300 expression was disrupted by a Short hairpin RNA. These results suggest that
curcumin might inhibit recruitment of RNAP II machinery through a mechanism

independent of the effect of curcumin on p300 or CBP HAT activity.

111

 

3. METHODS
Cell lines, viruses and treatment: HeLa (ATCC, Cat.# CCL-2) and Vero (ATCC, Cat.#
CCL-8l) cells obtained from the American Type Culture Collection were grown in
Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal
bovine serum (FBS, Gibco). The KOS strain of HSV-1 was used to infect HeLa cells at a
multiplicity of infection (MOI) of 1-5 pfu/cell, titered in Vero cells. Curcumin (Sigma,
Cat. # C1836) was dissolved in dimethyl sulfoxide (DMSO). Trichostatin A (TSA)
(Sigma, Cat. # T8552) was dissolved in ethanol. Cells in normal growth media were

treated with curcumin as indicated in figure legends.

Plasmids and transfection: Construction of the pSUPER-p300 plasmid has been
previously described (11). Briefly, a 64-mer doublestranded Oligonucleotide targeting
p300 was cloned into the pSUPER vector (OligoEngine) digested with Bgl II and Hind
III. The Oligonucleotide sequences are as follows, with bold font indicating the p300-
specific region: .

Forward primer: S’- GAT CCC CGT CTT GGC ATG GTA CAA GAT TCA AGA
GAT CTT GTA CCA TGC CAA GAC TTT TTG GAA A -3’

Reverse primer: 5’- AGC TTT TCC AAA AAG TCT TGG CAT GGT ACA AGA TCT
CTT GAA TCT TGT ACC ATG CCA AGA CGG G -3’

HeLa cells were transfected with 3 pg of the indicated plasmids using Lipofectamine

2000 (Invitrogen) according to manufacturer’s instructions.

112

 

Single-step growth curve: To determine growth curves of HSV-1 in the presence and
absence of curcumin, Vero cells were plated at a density of 1 x 106 cells per 60 mm tissue
culture plate. After 24 h, cells were pretreated for two hours with 20 M curcumin prior
to infection at a multiplicity of l pfu/cell. Duplicate samples were harvested at 4-h
intervals by dislodging cells into the overlying medium with a disposable scraper. The
cells were disrupted by sonication and insoluble cell debris was removed by

centrifugation. The titer of each sample was determined in triplicate by plaque assay.

Plaque assay: Plaque assays were performed by infecting nearly confluent 60 mm plates
of Vero cells in triplicate for each virus dilution. 100 pl of each dilution was added to
plates and plates were rocked every 15 minutes for 1 hour. After 1 hour, cells were
washed with DMEM lacking F BS and then overlaid with DMEM containing 2% FBS and
0.9% Sea Plaque Agarose. After 3 days, plates were stained with neutral red (0.2 mg/ml
in PBS) and incubated one more hour for visualization of plaques. Plaque sizes were
measured using a Nikon Eclipse TE300 microscope equipped with a Hamamatsu digital

camera, C4742-95, using OpenLab software.

ChIP assay: Chromatin immunoprecipitation (ChIP) assays were performed as described
previously (15). Briefly, HeLa cells were fixed with 1% formaldehyde and were collected
in a hypotonic buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5 % IGEPAL CA-630 with
protease inhibitors). Nuclei were released by dounce homogenizer, collected and
sonicated to obtain 200 to 500 bp DNA fragments. After preclearing with protein G-

agarose beads, IPs were performed overnight at 4°C. Antigen-antibody complexes were

113

precipitated by using protein G-agarose beads. Beads were washed and the protein DNA
complexes were eluted with 100 pl of 50 mM Tris-HCI (pH 8.0)-10 mM EDTA-1%
sodium dodecyl sulfate for 20 min at 65°C. Cross-links in DNA-protein complexes were
reversed by overnight incubation at 65°C and then precipitated with ethanol. DNA was
then further purified by Proteinase K digestion, phenolzchloroform (1 :1) extraction and
ethanol precipitation. DNA samples were resuspended in 75 ul of water and subjected to

quantitative real-time PCR analysis.

ChIP assays were performed using antibodies or antisera directed against VP16 (50),
RNA Pol II (8WG16; Covance), CBP (A-22; Santa Cruz Biotechnology), p300 (N-lS;
Santa Cruz Biotechnology), histone H3 acetylated at Lys9 (07-352, Upstate), histone H3
acetylated at Lysl4 (07-353, Upstate) and a C-terminal epitope of histone H3 (ab1791;

Abcam). Control IPS were performed using no antibodies.

RNA isolation, reverse transcription, Q-PCR: Total RNA was isolated by Trizol
according to manufacturer’s instructions. 1 ug RNA was reverse transcribed using the
Reverse Transcription System (Promega). Quantitative real-time PCR analysis for gene
expression and ChIP experiments was performed by SYBR Green assay using ABI 7500
real time PCR system (Applied Biosystems). The primer pair for the c-fos promoter was:
S’-GAACTGCGAAATGCTCACGAGATTA-3’ (forward) and S’-
AGTGTAAACGTCACGGGCTCAA-3’ (reverse). The primer pair for the c-fos ORF
was: 5’-TCAACGCGCAGGACTI‘CTG-3’ (forward) and S’-

GCAGTGACCGTGGGAATGA-3’ (reverse). The primer pair for p300 was: 5’-

114

CAATGAGATCCAAGGGGAGA-3’ (forward) and 5’-
ATGCATCT'ITCTTCCGCACT-3’ (reverse). The primer pair for CBP was: 5’-
GTGCTGGCTGAGACCCTAAC-3’ (forward) and 5’-
GGCTGTCCAAATGGACTTGT-3’ (reverse). Other primer pairs were previously

described (15, 41).

Western blot analysis: Histones from HeLa cells were isolated by acid-extraction. In
brief, nuclei were released by Triton extraction buffer (PBS containing 0.5% Triton X-
100 (v/v) and PMSF) and collected. Histones were then extracted using 0.2 N
hydrochloric acid. Acid soluble proteins were loaded onto a 15% sodium dodecyl sulfate-
polyacrylamide gel. Proteins were transferred to nitrocellulose membrane, blocked at
room temperature for 1 hr in 5% BSA/TTBS and proteins were identified using
antibodies specific to histone H3 (abl 791, Abcam) or H3 acetylated at Lys9 and Lysl4
(06-599, Upstate).

For the p300 western blot, total cell lysate was prepared from cells by RIPA buffer and
loaded onto a 6% sodium dodecyl sulfate-polyacrylamide gel. Proteins were transferred
to a nitrocellulose membrane and blocked at 4°C overnight in 5% non-fat dry milk-
TTBS. p300 was detected by a rabbit polyclonal antibody from Santa Cruz (SC-S 84).

GAPDH was detected by a mouse monoclonal antibody from Chemicon (MAB3 74).

115

4. RESULTS

Curcumin diminishes HSV-l infectivity and slows HSV-l replication:

A previous report indicated that curcumin could reduce the infectivity of HSV-2
virions when the chemical was added to virus preparations prior to infection (6). This
observation suggests that curcumin has a biochemical effect on the structure or integrity
of the virion itself. Given the evidence that curcumin can also inhibit a range of cellular
processes on which viral gene expression and replication might depend, we tested
whether the ability of cells to support HSV-1 infection and replication was altered by the
presence of curcumin. To address whether curcumin inhibits infection by HSV-1, we
performed plaque assays in cells that were treated with curcumin. The presence of
curcumin reduced the number of plaques on Vero cell monolayers by approximately
three-fold (Fig. 3.1A). The plaque Size, which reflects multiple rounds of infection, was
also affected by curcumin. After three days, the mean plaque Size in the presence of
curcumin was 0.62 (t 0.10) mm, whereas the mean plaque size in untreated cells was
0.73 (i 0.13) mm. The plaque size distributions in treated and untreated cells (Fig. 3.1B)
are significantly different (p 5 0.00005 by Wilcoxon rank-sum test). The plaque
morphology was also different in treated and untreated cells (Fig. 3.1C). In curcumin-
treated cells, although the cytopathic effects of late-stage HSV infection were obvious in
the middle of the plaque, the plaque was not cleared as in untreated cells. This effect
might also be an indication that the lytic infectious cycle is slowed in the presence of

curcumin. These results suggest that curcumin reduces the ability of cells to support HSV

infection, and that curcumin slows but does not totally block HSV-l replication.

116

We then tested further the effect of curcumin on viral replication kinetics and
production of infectious virions. Fig. 3.1D shows the results of a Single-step growth curve
assay indicating that production of infectious virus was diminished in cells treated with
curcumin. In mock-treated Vero cells, production of infectious progeny virus reached a
maximum of approximately 108 plaque-forming units per ml (pfu/ml) by 12 hours post-
infection (hpi). In the presence of curcumin, progeny virus titers were lower throughout
the infection period, but did increase steadily to a maximum of approximately 107 pfu/ml.
We conclude that treatment of cells with curcumin decreases the ability of cells to

support HSV-l infection and slows but does not block the viral replication cycle.

117

 

Figure 3.1: Effects of curcumin on HSV-1 plaque formation and viral replication. (A)
Plaque assays were performed in Vero cells pretreated (+) with 10 uM curcumin or
untreated (-) for 2 hours and infected with serial dilutions of a stock preparation of HSV-
1. 10 uM curcumin was also present in the viral inoculums and overlay agar media of
treated samples. Error bars represent the standard deviation among triplicate assays at
two viral dilutions. (B) Plaque sizes in the absence (open circles) or presence (closed
circles) of curcumin were measured by averaging randomly chosen vertical and
horizontal diameters from plaques. The numbers of plaques within various size ranges
(from less than 0.5 mm to greater than 0.9 mm) are shown. (C) Representative plaques
from plaque assays performed in the absence (left panel) and presence (right panel) of 10
uM curcumin. Scale bar: 100 um. (D) Single-step grth curves were performed in
Vero cells infected with HSV-1 at an MOI of 1 pfu/cell. Virus titers in samples collected
at 4 h intervals were assayed in triplicate on Vero cells. Closed circles represent
curcumin-treated samples; open circles represent parallel cultures of untreated cells.

118

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119

Curcumin inhibits IE gene expression without affecting the delivery of the
viral genome to the nucleus:

We have previously reported that the transcriptional coactivator and histone
acetyltransferase proteins p300 and CBP are recruited to IE gene promoters in a manner
dependent on the VP16 transcriptional activation domain (15). Curcumin can inhibit the
HAT activity of p300 and CBP but not of other mammalian coactivators such as PCAF
(3). We hypothesized that if the HAT activities of p300 and CBP are important for IE
gene expression and if curcumin inhibits that activity, then curcumin would inhibit IE
gene expression. Indeed, in HeLa cells treated with nontoxic levels of curcumin and
infected at a multiplicity of 1 pfu/cell, the steady-state mRNA levels for the IE genes
ICP4 and ICP27 were significantly reduced (Fig. 3.2A), whereas levels of CBP
transcripts (Fig.3.2A) and p300 transcripts (data not shown) were unchanged. This result
suggests that curcumin inhibits VP16-dependent transcription activation, potentially by
inhibiting p300/CBP HAT activity. Since curcumin accumulates in various cellular
membranes (20), we were concerned that it might interfere with viral entry or delivery of
the viral genome to the nucleus, which would result in diminished IE gene expression.
This concern was alleviated by assaying viral DNA levels in the nuclear fi'actions of
curcumin-treated and mock-treated cells. Quantitative PCR results shown in Fig. 3.2B
Show no difference in viral DNA levels between treated and untreated cells. We
conclude that curcumin treatment does not disrupt the delivery of viral genomes to the
infected cell nucleus, and infer that curcumin has a more direct role in inhibiting IE gene

transcription.

120

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Figure 3.2: Effects of curcumin on HSV-1 IE gene expression. (A) HeLa cells were
pretreated with 20 uM curcumin (+, black bars) or DMSO only (-, open bars) for 3 hours
and then infected with HSV-1 at an MOI of 1 pfu/cell. Total RNA was isolated at 2 hpi
and gene expression was analyzed by Q-RT-PCR, normalized against 18S rRNA levels.
Mean values from three experiments for each gene tested are shown relative to values
obtained from untreated cells; error bars represent standard deviations. (B) HeLa cells
were infected with HSV-1 in the presence (+, black bars) or absence (-, open bars) of 20
uM curcumin. Aliquots of nuclear extracts (as used in ChIP assays, Fig. 3.3) were
analyzed by Q-PCR. The amount of viral DNA (represented by the ICP27 gene
promoter) was normalized against cellular DNA (represented by the U3 snRNA
promoter). Error bars represent the standard deviation among five independent
experiments.

121

Curcumin decreases RNAP II occupancy on IE gene promoters and ORFS
but does not affect recruitment of VP16:

One way that curcumin might block IE gene expression is by decreasing the
recruitment of VP16 to IE gene promoters. Alternatively, curcumin might affect
recruitment of RNAP 11 without affecting VP16 occupancy. To test these hypotheses, we
performed chromatin immunoprecipitation (ChIP) assays to probe the presence of VP16
and RNAP II at IE genes in HeLa cells infected at an moi of 5 pfu/cell. Curcumin had no
substantial effect on the recruitment of VP16 to the ICPO or ICP27 promoters (Fig. 3.3A).
The U3 snRNA gene promoter served as a negative control, showing that VP16
occupancy as detected by ChIP assays was specific to viral IE promoters, as previously
shown (15). In contrast, curcumin caused a Significant decrease in the occupancy of
RNAP II on both the promoter and transcribed region (ORF) of ICP27 (Fig. 3.3 B).
Similar decreases in RN AP 11 occupancy were observed for the ICPO gene promoter (data
not Shown). The modest effect of curcumin on the presence of RN APII on the U3 snRN A
promoter was not observed in other replicates of this experiment (data not shown). We
conclude that curcumin decreases VP16-dependent recruitment of RNAP II and hence IE

gene expression.

122

 

 

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Figure 3.3: Curcumin decreases occupancy of RNA polymerase II but not of VP16 at IE
genes. HeLa cells treated with 20 11M curcumin (+, black bars) or DMSO only (-, open
bars) were infected with HSV-1 at an MOI of 5 pfu/cell. Chromatin immunoprecipitation
from sonicated nuclear extracts was performed using antibodies against VP16 (A) or
RNA pol II (B). Samples were analyzed by Q-PCR using primers specific for the ICPO
promoter, ICP27 promoter, ICP27 ORF or the cellular U3 snRNA promoter. Aliquots of
samples prior to immunoprecipitation (1%, 0.3%, 0.1%, 0.04% input) were used to create
a stande curve of input DNA. This curve was used to determine the relative amounts of
a given DNA fragment in immunoprecipitated or control (no antibody) samples. The
values for no-antibody controls were subtracted from the values for corresponding 1?
samples. Error bars represent the range between technical duplicates for a representative
experiment.

123

Curcumin has no significant effect on H3, AcH3, p300 and CBP occupancy
on IE gene promoters and ORFs:

One of the potential ways that curcumin might inhibit RNAP II recruitment is by
inhibiting p300/CBP HAT activity (3, 34), thus permitting formation of transcriptionally-
repressive chromatin on viral DNA. We first tested whether the levels of curcumin (20
pM) that inhibit viral gene expression also affect total acetylation levels of histone H3.
An immunoblot using an antibody specific to acetylated Lys9 and Lysl4 of H3 revealed
no significant change in total histone H3 acetylation after three hours, five hours, or
overnight incubation in 20 pM curcumin (Fig. 3.4A). Those times correspond to
beginning and end of viral infection, respectively, for the viral gene expression analysis
indicated in Fig. 3.2A. A decrease in histone H3 acetylation was observed only after
overnight incubation in the presence of higher curcumin concentrations (Fig. 3.4A). We
infer that, under the conditions used in viral gene expression assays, curcumin does not
significantly inhibit p300/CBP HAT activity.

We have previously shown that HSV-l IE gene promoters are relatively devoid of
histone H3 at 2 hpi, but that H3 and acetylated H3 can be found at IE gene ORFS and also
at DE and L gene promoters at that time (15). However, during infection with a mutant
virus (strain RPS) lacking the VP16 activation domain, H3 did associate with IE
promoters, but acetylated H3 levels at IE ORF and DE or L gene promoters were
reduced. On that basis, we hypothesized that p300/CBP HAT activity might be required
for covalent modification and subsequent removal of histones from IE gene promoters, to
allow recognition of those promoters by RNAP 11. That model led to the prediction that

curcumin, by inhibiting p300/CBP HAT activity, would diminish levels of acetylated

124

 

histones at IE gene promoters and ORF S, and would increase the level of H3 at IE gene
promoters, as observed during RPS infection (15).

ChIP assays were performed using antibodies that recognize an H3 carboxyl-
terminal epitope unaffected by known modifications or antibodies specific to H3
acetylated at either Lys9 or Lysl4. Contrary to our hypothesis, we observed no
significant increase in H3 occupancy (Fig. 3.4B) and no significant decrease in K9-
acetylated H3 (Fig. 3.4C) or K14-acetylated H3 (Fig. 3.4D) occupancy on the ICP27
promoter or ORF. We conclude that the inhibition of IE gene expression caused by
curcumin is not the result of changes in histone occupancy or acetylation at IE gene
promoters, and thus the HAT activity of CBP and p300 is not likely to be critical for viral
IE gene expression.

A recent report has suggested that relatively high concentrations of curcumin can
inhibit the recruitment of p300 to promoter DNA fragments in vitro (4). Thus, we
considered the possibility that although their HAT activity per se might not be required,
p300 and CBP might act as scaffolding proteins at IE gene promoters for the recruitment
of other transcription factors. We used ChIP assays to test whether curcumin inhibits the
recruitment of p300 and CBP to IE gene promoters. The results (Fig. 3.4E,F) indicate
that curcumin did not block the recruitment of p300 or CBP to IE gene promoters nor to
the c-fos promoter, which was previously Shown to be dependent on p300/CBP (19, 43).
Instead, we observed a modest increase in recruitment of p300 and CBP at the ICP27
promoter. One potential explanation for this increase is that curcumin can inhibit the
autoacetylation of p300 or CBP and hence inhibit the dissociation of CBP from the IE

gene promoter, which would in turn prevent the recruitment of RNAP II. Taken together,

125

these results suggest that curcumin affects recruitment of RNAP II to the HSV-1 genome
but not to cellular control gene promoters, and that this effect is independent of the

effects of curcumin on p300 and CBP HAT activity.

126

Figure 3.4: Curcumin does not affect histone H3, p300, or CBP occupancy, nor H3
acetylation, at IE genes. (A) HeLa cells were treated with curcumin (concentrations
given in uM) for various times in the presence of 1.4 pM trichostatin A. Acid extracted
histones were analyzed by immunoblotting using antibodies directed against H3
acetylated at Lys9 and Lysl4 (top panel) or a carboxyl-terrninal epitope of H3 (bottom
panel). (B-F). Chromatin immunoprecipitation was performed as described for Fig. 3.3,
but using antibodies against the H3 carboxyl-terminal domain (B), H3 acetylated at Lys9
(C), H3 acetylated at Lysl4 (D), the coactivator p300 (E), or the coactivator CBP (F).
The %input values in B-D were further normalized to U3 snRN A promoter as a means to
control immunoprecipitation efficiency. Since the p300/CBP IP efficiencies were low,
this data is represented as fold enrichment over no antibody control. Error bars represent
the standard deviation among Six (for ICP27 promoter) and four (for c-fos promoter)
independent experiments (D, E). p-values in (D,E) were determined by Student’s-t-test.

127

 

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Diminished expression of p300 does not affect IE gene expression:

In order to validate the conclusion that curcumin inhibits IE gene expression
independent of p300 HAT activity, we have analyzed IE gene expression when p300
expression was diminished by RNA interference. Transfection of a plasmid expressing a
Short hairpin RNA (shRNA) specific for p300 resulted in efficient disruption of p300
expression assessed as either protein (Fig. 3.5A) or as mRNA (Fig. 3.5B). The disruption
was specific for p300, since CBP expression was not affected (Fig. 3.5B). However, viral
IE gene expression was not significantly affected (Fig. 3.5C), suggesting that p300 is not
required for HSV-1 IE gene expression. This result also argues against the possible role
of p300 as a scaffolding protein on IE gene promoters. Another possible explanation is
that p300 and CBP may act redundantly on viral IE gene promoters and therefore
knocking down only p300 does not affect IE gene expression. Another possible
explanation is that p300 and CBP may act redundantly on viral IE gene promoters and
therefore knocking down only p300 does not affect IE gene expression. We are currently
working on a more detailed analysis of coactivator disruption, which will be the subject
of another manuscript. Our preliminary results suggest that neither p300 nor CBP are

essential for IE gene expression (Kutluay, et al., manuscript in preparation).

129

 

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Figure 3.5: Knocking down p300 expression does not affect HSV-1 IE gene expression.
HeLa cells were transfected with 3 pg of pSUPER (empty) or pSUPER-p300 plasmid
that expresses a shRNA targeting p300. 48 hours post-transfection cells were infected
with HSV-l at an MOI of 5 pfu/cell and total protein and RNA were isolated. p300
knockdown efficiency was analyzed by immunoblotting (A) and Q-RT-PCR (B). IE gene
expression was analyzed by Q-RT-PCR as indicated in previous figure legends (C). Data
represents the average of three independent experiments done at least in biological
duplicates. Error bars represent the standard deviation.

130

 

5. DISCUSSION

This study was performed to assess whether curcumin would act as an antiviral
agent for HSV-1 infection, based on the hypothesis that curcumin could prevent IE gene
expression by inhibiting the p300 and CBP HATS (3) that are recruited to IE gene
promoters during lytic infection (15). Our results demonstrate that curcumin-treated cells
are indeed diminished in their ability to support HSV-1 infection and replication. The
presence of curcumin did not block the delivery of the viral genome to the nucleus,
indicating that binding, entry, and intracellular transit of the virus are not substantially
affected by curcumin. We did observe a significant decrease in expression of viral IE
genes, corresponding to diminished recruitment of RNAP II to IE gene promoters. These
outcomes are all consistent with the hypothesis that curcumin specifically affects viral IE
gene expression.

However, additional observations suggest that the antiviral effects of curcumin do
not arise through the mechanism initially proposed. The effects of curcumin on IE gene
expression were evident using concentrations and times much less than those required for
significant inhibition of overall histone acetylation. We expected that curcumin, as an
inhibitor of p300 and CBP HAT activity would result in diminished histone acetylation
and increased histone H3 occupancy at IE gene promoters, but those effects were not
observed. Moreover, disruption of p300 protein expression had no significant effect on IE
gene expression. We conclude that the HAT activity of p300 and CBP is not essential for
histone clearance from HSV IE gene promoters or for IE gene expression, but that
curcumin blocks other, yet unknown steps that are required for recruitment of RNAP II

and subsequent transcription of IE genes. We cannot yet exclude the possibility that other

131

 

 

 

HATS such as PCAF and hGCNS may act redundantly on viral genes, masking any
curcumin-dependent changes in histone acetylation (9).

Several potential mechanisms whereby curcumin blocks IE gene expression can
be eliminated based on additional ChIP assays. The recruitment of the virion-bome
activator protein VP16 to IE promoters was not affected by curcumin. Although a prior
report suggested that curcumin can inhibit recruitment of p300 to target gene promoters
by the chimeric activator protein Gal4-VP16 in an in vitro experiment (4), we observed
no effect of curcumin on p300 or CBP occupancy on IE gene promoters. One possible
reason for this discrepancy might be the high concentrations of curcumin (300 pM) used
in the in vitro system (4), whereas we used 10-20 pM curcumin because of cytotoxicity
observed at higher concentrations and long incubation periods.

Our observation that the occupancy of histone H3 and acetylated H3 on IE gene
promoters and ORFS did not change upon curcumin treatment leads to the question of
why the dramatic decrease in viral gene expression does not correspond to an increase in
histone occupancy on the viral genome. Since histones are not packaged with the HSV-1
genome in virion particles (12), one possibility is that the level of histone deposition on
the viral genome at 2 hours post-infection is still relatively low compared to cellular
genes and hence the histones do not create a substantial barrier for transcription. Another
possibility is that the VP16 activation domain recruits other coactivators such as the
chromatin remodeling enzymes Brm and Brg-l (15), independent of the HAT activities
of CBP and p300, to keep the viral genome free of histones. In support of the latter
hypothesis, VP16 AD has been recently Shown to recruit the yeast remodeling complex

SWI/SNF to mono- and dinucleosomal arrays to catalyze the eviction of nucleosomes by

132

 

SW1/SNF (14).These hypotheses will be tested in the context of viral infection in future
experiments.

We note that the relatively modest effects of curcumin on the viral infection and
replication (Fig. 3.1) do not seem to reflect the dramatic reduction in IE gene expression
(Fig. 3.2A) and in RNAP II recruitment to IE promoters (Fig. 3.3B). We suspect that this
discrepancy may be due to the decay of the biological activity of curcumin when exposed
to light or to cell culture media (5, S4) in the longer experiments probing viral infection
and replication, relative to the shorter experiments used for IE gene expression and ChIP
assays.

Others have reported that virions of HSV-2 were rendered less infectious by
exposure to curcumin prior to infection of HeLa cells or of mouse genitalia (6). To date,
no biochemical mechanism for this virucidal effect has been established. In contrast, the
work presented here indicates that curcumin can inhibit viral gene expression and thus
replication through mechanisms operative within the infected cells. This is especially
evident in our observations that curcumin did not affect delivery of viral DNA to the
nucleus, but had a dramatic effect on IE gene expression (Fig. 3.2). Nonetheless, we
cannot fiilly exclude the possibility that the virucidal effects of curcumin might contribute
to the diminished replication and plaque formation evident in Fig. 3.1.

Although curcumin has been widely studied in the context of cancer and
apoptosis, the current literature does not clearly define the effects of curcumin on
RNAPII machinery or transcription factors. One report has shown that curcumin inhibits
certain cellular differentiation events by triggering protease-dependent degradation of the

transcription factor AP-l (2). Although our results cannot directly exclude the possibility

133

 

 

that VP16 is degraded similarly, our ChIP data suggests that more or less equivalent
amounts of VP16 are immunoprecipitated with IE gene promoters and hence the
mechanism whereby curcumin inhibits viral IE gene expression is unlikely to be
mediated by degradation of VP16. The ChIP results also suggest that curcumin does not
prevent the formation of the DNA-binding protein complex comprising VP16, Oct-1 and
HCF. Since VP16 itself cannot bind to DNA with high affinity (1, 26, 35) if curcumin
was preventing the interaction between VP16, Oct-1 and HCF, then less VP16 would
have been immunoprecipitated to IE gene promoters in the presence of curcumin.
Curcumin is a potent compound with various biological properties. We have
shown that curcumin significantly affects HSV-1 IE gene expression which thereby
diminishes the ability of the virus to launch the lytic infectious cycle. Whether curcumin
can be used as an anti-HSV-l therapeutic agent will likely be limited by its low
bioavailability and high degradation rates when exposed to light. Moreover it is still not
known how curcumin is metabolized in vivo and how these by-products might affect
cellular processes. Therefore understanding the fate of curcumin in vivo is key to
developing curcumin as an alternative drug for HSV-1 treatment. Hence, our results can
be considered as an early step in elucidating the molecular basis of the antiviral activities

of curcumin.

134

6. ACKNOWLEDGEMENTS

This work was supported by the Department of Biochemistry and Molecular Biology at
Michigan State University and by the Van Andel Research Institute. SK was supported
by a Special fellowship from the College of Natural Science at Michigan State University
and by a predoctoral fellowship from the American Heart Association. JD was supported
in part by summer undergraduate research fellowships from the American Society for
Microbiology. We thank Dan Ducat and Dr. F ranciso Herrera for preparing the pSUPER-
p300 construct. We also thank Dr. Eric Xu, Dr. Xu Lu, and Dr. Francisco Herrera for

thoughtful comments on the manuscript.

135

 

10.

11.

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CHAPTER FOUR

Kutluay, Sebla, B., Triezenberg, Steven, J. (2009). Regulation of Histone Deposition on
the Herpes Simplex Virus Type 1 Genome During Lytic Infection. Journal of Virology,

 

141

 

Chapter 4

MECHANISMS OF HISTONE DEPLETION FROM THE VIRAL
GENOME

1. ABSTRACT

During lytic infection by herpes simplex virus type 1 (HSV-1), histones are
present at relatively low levels on the viral genome. However, the mechanisms that
account for such low levels—how histone deposition on the viral genome is blocked or
how histones are removed from the genome—are not yet defined. In this study we Show
that histone occupancy on the viral genome gradually increased with time when
transcription of the viral immediate-early (IE) genes was inhibited either by deletion of
the VP16 activation domain or by chemical inhibition of RNA polymerase II (RNAP II).
Inhibition of IE protein synthesis by cycloheximide did not affect histone occupancy on
most IE promoters and coding regions, but did cause an increase at delayed early and late
gene promoters. IE gene transcription from HSV-1 genomes associated with high levels
of histones was stimulated by superinfection with HSV-2 without altering histone
occupancy or covalent histone modifications at IE gene promoters. Moreover, RNAP II
and histones co-occupied the viral genome in this context, indicating that RNAP 11 does
not preferentially associate with viral genomes that are devoid of histones. These results
suggest that during lytic infection, VP16, RNAP II, and IE proteins may all contribute to
the low levels of histones on the viral genome, and yet the dearth of histones is neither a
prerequisite for nor a necessary result of VP16-dependent transcription of nucleosomal

viral genomes.

142

 

2. INTRODUCTION

During lytic infection of mammalian cells by herpes simplex virus type 1
(HSV-1), virion protein 16 (V P16) triggers the cascade of viral gene expression by
stimulating the transcription of immediate early (IE) genes (5). VP16 binds to the cis-
regulatory sequences on viral IE gene promoters as part of a protein complex that also
includes two host cell proteins, Oct-1 and HCF (67). VP16, through its activation domain
(AD), then interacts with various general transcription factors or coactivators and recruits
the host RNA polymerase II (RNAP II) machinery (13, 23, 27, 42, 68). In turn, some of
the IE proteins regulate expression of the delayed early (DE) and late (L) genes,
completing the viral gene expression cascade that results in production of infectious
virions.

In eukaryotes, the packaging of DNA by histone proteins presents a significant
impediment to the transcriptional machinery (41). This barrier can be overcome by
activator-mediated recruitment of transcriptional coactivators that either covalently
modify histones (29, 41) or remodel the position of nucleosomes along DNA (11, 56).
Many covalent modifications of histones have been identified, including lysine
acetylation and methylation (29, 41); the former is generally associated with active
transcription and the latter marks either active or inactive transcription depending on
which lysine residue is methylated.

Unlike the small DNA tumor viruses of the polyomavirus and papillomavirus
families, the large (152 kbp) genome of HSV-1 is packaged in the virion nucleocapsid
not with histones but with the polyamine spermine (7, 12, 50, 53). Long-standing

evidence suggests that during lytic infection the HSV-1 genome is mainly non-

143

nucleosomal (39, 40, 46), whereas during latency the viral genome is associated with
evenly Spaced nucleosomes (10). In support of these earlier findings, chromatin
immunoprecipitation (ChIP) assays have indicated that histones, typically represented by
histone H3, are present on the viral genome during lytic infection, albeit at relatively
lower levels than on cellular genes (20, 21, 26, 35, 50). Furthermore, active transcription
marks such as histone H3 acetylated on lysine 9 and lysine 14 (H3 K9/K14ac) or
trimethylated on lysine 4 (H3K4me3) are also associated with the viral genome during
lytic infection (20, 21, 26, 35). In contrast, during latent or quiescent infections, histones
associated with the viral genome carry modifications typical of heterochromatin, such as
histone H3 trimethylated on lysine 9 (H3K9me3) (8, 66).

An exception to this pattern is the latency-associated transcript (LAT) gene, which
during latency is associated with H3 K9/K14ac, indicative of its transcriptionally active
status (31, 32, 49). Moreover, upon reactivation from latency, lytic genes become
associated with acetylated histone H3, and the LAT region becomes depleted of that mark
(1, 49). Together, these observations suggest that regulation of chromatin might be an
important mechanism for the switch between lytic and latent infections (28).

Although histone H3 occupancy is presumed to represent the existence of the
nucleosome core particle on the viral genome, whether other core histones are present on
the HSV-1 genome during lytic infection has not been established. This question
becomes more compelling in light of the evidence that histone H2A/H2B dimers can be
removed from nucleosome core particles, resulting in the presence of “hexasomes”, at
actively transcribed genes (19, 33, 58). In addition, the mechanisms regulating histone

levels on the viral genome during lytic infection have not yet been defined. One key issue

144

 

 

is whether histone deposition is inhibited or deposited histones are removed. Hints of
potential mechanisms have arisen in several recent reports. For instance, an IE protein,
ICPO, blocks the silencing of HSV-1 DNA by dissociating the histone deacetylase 1 from
the REST/CoREST/HDAC repressor complex (14, 15). During lytic infection with a
mutant virus that does not express ICPO, histone H3 occupancy on the viral genome
increased and the fraction of acetylated histone H3 decreased, suggesting that the
disruption of the REST/CoREST/HDAC complex by ICPO might be relevant (6).
Similarly, the tegument protein VP22 was suggested to block nucleosome assembly on
the viral DNA (64), but no data yet indicate whether or how histone occupancy on the
viral genome changes in the absence of VP22 during HSV-1 lytic infection.

Another factor that may mediate histone levels on the viral genome during lytic
infection is VP16. We have previously shown that during lytic infection with a mutant
virus that lacks the activation domain of VP16 (strain RPS), higher levels of histone H3
associate with RPS genomes than with wild-type genomes (20). Moreover, during RPS
infections, transcriptional coactivators—such as p300 and CBP histone acetyl
transferases (HATS) or BRM and Brg-l remodeling enzymes—are not efficiently
recruited to IE gene promoters (20). This observation is consistent with prior knowledge
about the VP16 AD in artificial experimental contexts, in which VP16 physically
interacts with and recruits transcriptional coactivators—such as HATS p300/CBP (3, 17,
24, 34, 62, 65), PCAF (65), and GCN5 (22, 37, 59, 62, 63), as well as ATP-dependent
chromatin remodeling enzymes (16, 43, 47, 48)—to potentiate transcription from
nucleosomal templates. However, the hypothesis that the transcriptional coactivators

recruited by VP16 are required for modifying and removing the histones fiom the viral

145

genome, and thereby required for IE gene expression, is contradicted by our recent report
that disruption of coactivator expression did not diminish IE gene expression during
infection of cultured cells (36). Thus, the impact of histones and chromatin on viral gene
expression remains incompletely defined.

In the present report, we show that during lytic infection of cultured cells, all four
core histones associate with the viral genome. In the absence of VP16 transcriptional
activation (i.e., during infection by the RPS mutant virus), histone deposition throughout
the viral genome increased gradually but dramatically to levels approaching that on
cellular genes. Inhibition of RNAP II-mediated transcription by actinomycin D also
increased the histone occupancy on the viral genome in a temporal pattern similar to that
of RPS infections. In contrast, inhibition of IE protein expression by cycloheximide had
no significant effect on histone association with most of the actively transcribed IE gene
promoters and coding regions, but did increase histone occupancy on DE and L gene
promoters. To address whether VP16 can stimulate the removal of histones that are
already deposited on the viral genome, we asked whether providing wild-type VP16 in
trans (by HSV-2 superinfection) results in histone depletion from RPS IE gene
promoters. Surprisingly, although HSV-2 superinfection stimulated IE gene expression
from the RPS genome, it did not lead to depletion of histones from IE genes. In addition,
active transcription marks, such as H3 K9/K14ac or H3K4me3, did not increase on the
RPS IE gene promoters upon HSV-2 superinfection. Sequential chromatin
immunoprecipitation (Seq-ChIP) experiments indicated that RNAP II and histone H3 co-
occupy RPS genomes upon HSV-2 superinfection at a level similar to that of a

constitutively expressed house-keeping gene, indicating that RNAP 11 does not

146

preferentially associate with histone-free viral genomes.

Taken together, our results suggest that the low level of histone occupancy on the
viral genome during lytic infection is the result of a complex process that involves VP16,
active transcription by RN AP 11, and IE proteins. However, histone removal or covalent
modification of histones may not be necessary for the VP16-dependent transcription of IE

genes from viral genomes heavily associated with histones.

147

3. METHODS
Cell lines and viruses: HeLa (ATCC# CCL-2) cells were grown in Dulbecco’s modified
Eagle’s medium (Invitrogen) containing 110 mg/l sodium pyruvate and 10% fetal bovine
serum(1nvitrogen). In some experiments, cycloheximide (Sigma) or actinomycin D
(Sigma) were added to the cell culture medium prior to and during infection, as indicated
in figure legends. The RPS strain of HSV-1, which lacks sequences encoding the
activation domain of VP16, has been previously described (60). The RPS and wild-type

KOS strains of HSV-1 and G strain of HSV-2 were prepared and titered in Vero cells.

Gene expression assays and Q-RT-PCRs: Total cellular RNA was isolated using Trizol
reagent (Invitrogen). Total RNA was reverse-transcribed by random primers using a
reverse transcription system (Promega). The synthesized cDNA was used as template in
quantitative real-time PCR analysis using SYBR Green Master Mix (Roche) and the ABI
7500 Real-Time PCR System (Applied Biosystems). Gene expression was first
normalized against 18S rRNA and then to proper controls by the Z—MCt method. For
chromatin immunoprecipitation assays, data were analyzed using the standard curve
method as explained in the following section. Primer sequences for PCRS spanning the
ICP27 promoter and gC promoter are as follows:
ICP27 promoter: (F) 5'-TGGTGTCTGATTGGTCCTTG and

(R) 5'-CGGGTGGTGGATGTCCTTAT
gC promoter: (F) S'-TCGGGCGATTGATATATTTTT and

(R) 5'-TGTCCCCTTCCGGAATI‘TAT

Other primer pairs used in this study have been previously defined (20, 51).

148

Chromatin immunoprecipitation and sequential-chromatin immunoprecipitation:
Chromatin immunoprecipitation was performed as previously described (20). In
summary, confluent plates of HeLa cells were infected in the absence or presence of
actinomycin D or cycloheximide as indicated in figure legends. Infections were stopped
by addition of formaldehyde to the cell culture plate at a final concentration of 1%. After
cells were resuspended in a hypotonic buffer, nuclei were released by Dounce
homogenization in order to minimize the background signals from cytoplasmic capsids or
membrane-bound virions. Nuclei were collected by centrifugation and then disrupted by
sonication using a Branson Digital Sonifier-450 to obtain 200-1000 bp DNA fragments.
Protein-DNA complexes were immunoprecipitated using 5-10 pg of antibodies against
histone H2A (Abcam, abl8255), histone H2B (Abcam, ab1790), histone H3 (Abcam,
ab1791), histone H4 (Upstate, 05-858), RNA polymerase II (Covance, 8WG16), or VP16
[ref (61)]. Protein-DNA complexes were collected by Protein G-agarose beads
(Invitrogen). After several washes, the protein-DNA complexes were eluted and reverse-
crosslinked overnight at 65 °C in the presence of 200 mM NaCl and 10 pg RNaseA.
Samples were then precipitated with ethanol, digested with proteinase K (Roche) at 42 °C
for 2 h, and purified with Qiagen spin columns using the gel extraction protocol. The
presence of viral and cellular DNA fragments in the immunoprecipitated material was
analyzed by quantitative real-time PCR using SYBR Green Master Mix (Roche) and ABI
7500 Real-Time PCR system (Applied Biosystems). A standard curve using serial 3-fold
dilutions of input samples (1%, 0.3%, 0.1%, or 0.04%) was produced to quantitate the
signals from immunoprecipitation samples. Background Signals obtained from

immunoprecipitation reactions performed in the absence of antibodies (no antibody

149

control) were subtracted from the signals obtained fi'om immunoprecipitation samples
[referred as “% input (IP-noab)”]. When necessary, data was firrther normalized against a
cellular control gene (interferon-[3 promoter or U3 snRNA promoter) by dividing the “%
input (IP-noab)” value for the viral DNA to that of the cellular DNA in order to account
for the differences in immunoprecipitation efficiencies.

For sequential ChIP assays, after the first immunoprecipitation, protein-DNA
complexes were eluted in 100 pl elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA,
1% SDS) by incubating at 65 °C for 15 min. The eluates were then subjected to the
second immunoprecipitation as indicated above, whereby 30-45 pl of eluate was used as
input in each immunoprecipitation reaction. The rest of the procedure is the same as the

ChIP protocol indicated above.

150

 

4. RESULTS

Absence of the activation domain of VP16 causes an increase in histone
occupancy on the HS V-I genome during lytic infection.

We have previously reported that during lytic infection by wild-type HSV-1, the
IE gene promoters are relatively free of histones as represented by H3 (20). We have
also shown that at immediate early times during infection by strain RPS (lacking the
VP16 activation domain), the HSV-1 genome more abundantly associates with histone
H3 but lacks acetylated histone H3, an active transcription mark (20). In order to get a
more dynamic and quantitative picture of histone deposition on the viral genome in the
presence or absence of the VP16 AD, we have compared histone occupancy on the RPS
and KOS (wild-type) genomes at different stages of infection using ChIP assays coupled
with Q-PCR. Parallel plates of HeLa cells were infected with KOS or RPS viruses at
multiplicities of infection such that similar numbers of viral genomes entered cells at the
beginning of infection, as shown by quantitative PCR assays of infected cell nuclear
extracts (Fig. 4.1). At 2, 4, and 6 hours post-infection (hpi), infections were stopped by
formaldehyde cross-linking and the occupancy of histone H2A, H2B, H3, H4, and RNAP

II on viral genes was analyzed by ChIP.

151

 

 

100 El RPS I KOS

=1:
3
o 10
O
9
E 1
2

0.1

2 4 6
hours post infection

Figure 4.1: Similar numbers of RPS and KOS viruses enter HeLa cells and RPS viruses
are debilitated in replication. The relative amount of viral DNA in RPS and KOS
infections was compared using the input standard curves from ChIP assays presented in
Figure 4.2. The amount of viral DNA (represented by the ICP27 gene promoter) was
normalized against cellular DNA (represented by the U3 snRN A promoter).

152

 

As indicated in Figure 4.2A, occupancy of histone H2A on the RPS genome at 2
hpi was higher than on the K08 genome at several viral gene fragments such as IE
promoters and ORFS (Fig. 4.2A, left panel) as well as at DE and L promoters (Fig. 4.2A,
right panel). Histone H2A occupancy on the RPS genome continued to increase at 4 and 6
hpi up to a point comparable to that of the cellular gene (i.e., an input ratio of
approximately 1). Similar patterns were observed for histone HZB (Fig. 4.2B), H3 (Fig.
4.2C), and H4 (Fig. 4.2D). Occupancy by all core histones throughout the viral genome
increased prominently but gradually in RPS infections, whereas in KOS infections
histone occupancy either stayed same or decreased during the later stages of lytic
infection. As expected, recruitment of RNAP II to IE promoters, and as a consequence to
DE and L gene promoters, was severely impaired in RPS infections (Fig. 4.2E). These
results together suggest that the VP16 AD contributes to keeping the viral genome free of
nucleosomes.

This effect could conceivably be mediated directly by the VP16 AD, i.e., by
recruiting transcriptional coactivators that modify and remodel the chromatin structure.
However, we have recently indicated that transcriptional coactivators recruited by VP16
do not contribute significantly to IE gene expression during lytic infection of human
foreskin fibroblasts (36). Alternatively, VP16 may indirectly regulate chromatin, e.g., by
stimulating the expression of IE genes that may in turn contribute to lower histone levels
on the viral genome. This concept is supported by the observation that histone occupancy
on delayed early (tk) and late (gC) gene promoters, where VP16 does not bind directly,
also increased substantially in RPS infections with a kinetics Similar to that on IE gene

promoters (Fig. 4.2). A further possibility is that active transcription by RNAP 11 itself

153

 

 

may prevent histone deposition, a mechanism that has been suggested for several actively
transcribed genes (4, 19, 33, 55). These last two possibilities are addressed in the next

sections.

 

154

Figure 4.2: Core histone occupancy on the viral genome increases in RPS lytic infections.
HeLa cells were infected with KOS (K) or RPS (R) strains of HSV-1 at an MOI of l
pfu/cell or 0.005 pfu/cell, respectively. Chromatin immunoprecipitation was performed to
detect the occupancy of histones H2A (A), HZB (B), H3 (C), H4 (D), and RNA
polymerase II (E) on ICP27, ICPO, tk, and gC promoters (pro) and on the ICP27 ORF at
2, 4 and 6 h post-infection. The data were analyzed as explained in Materials and
Methods. The graphs Show the results of a representative experiment; similar results were
observed in replicate experiments.

155

 

 

A. Histone H2A ChIP

   
 
 
  

2 4 6
hours post infection

input ratio (viraiIiFN-Bp)
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'l" iCP21pro (K)
+iCP27pro (R)
4- iCPOpro (K)
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‘-°- ICP27ORF (R)

 

 

 

'1'- iCP27pro (K)
+ ICme (R)
+|CP°PT° (K)
+ iCPOpro (R)

. -0- K:P21ORF (K)

 

r-o-icP270RF (R)

 

 

 

 

-°- lCPZ'Ipro (K)
+ iCP27pro (R)
1+ iCPOpro (K)
+ iCPOpro (R)
-0-lCP21ORF (K)

 

-0- lCP27ORF (R)

 

 

156

Input ratio (viraiIiFN-Bp)
e
' b

2 4 6
hours post infection

O‘NU.

input ratio (viraiIiFN-Bp)
p o o o 9

hours post infection

a

'2? 0.0

1L

5

g 0.4

a.

802 .._-g::=a
e

i

5

2 4
hours post infection

input ratio (viraiIlFN-Bp)
O
. . '-

30.0
% ll
\

0.4 ' --o
E. 'F‘~
s ‘~
g 0.2 I’ \
i
.E 0.0

hours post infection

 

 

-¢- tirpro (K)
+ tirpro (R)
+ scnro (K)
"' 00900 (R)

 

 

 

 

-¢- tkpro (K)
+ “(W0 (R)
"' 061m 00
'°' 06m (Ri

 

 

 

 

-¢- tirpro (K)
-I- tkpro (R)
+ 061m (K)
+ 061m (R)

 

 

 

 

-- tltpro (K)
+ tirpro (R)
-°- score 00
'°' 00910 (R)

 

 

 

 

-°- them 00
*- mm (R)
-°- 009m (K)
+ score (R)

 

 

 

 

 

 

 

 

Inhibition of transcription increases histone occupancy on the viral genome
during lytic infection.

Evidence in yeast has indicated that histones can be depleted from actively
transcribed regions (19, 30, 33, 38, 58). Since the increase in histone occupancy on the
viral genome in RPS infections might be dependent on the inability to recruit the RN AP
II machinery (Fig. 4.2E), we studied histone deposition on the viral genome during KOS
infections under circumstances in which RNAP II-mediated transcription was blocked.

To this end, HeLa cells were pretreated with actinomycin D and subsequently infected

 

with KOS in the presence of actinomycin D. Infections in mock-treated cells were carried
out in parallel. At 2, 4, and 6 hpi, infections were stopped by formaldehyde cross-linking
and ChIP was performed to assay the presence of core histones, RNAP II, and VP16 on
the viral genome.

As indicated in Figure 4.3A, the occupancy of histone H2A was not substantially
higher on viral genes at 2 hpi in the presence of actinomycin D than in its absence.
Similar to RPS infections, at later times in infection the H2A Occupancy throughout the
viral genome increased in the presence of actinomycin D and reached a level about half
that of the cellular gene.

Similarly, the occupancy of histones HZB (Fig. 4.3B), H3 (Fig. 4.3C), and H4
(data not shown) on the viral genome increased in the presence of actinomycin D with a
kinetics similar to that observed in RPS infections, although at 2 hpi, histone deposition
on the viral genome was slightly less than that observed in RPS infections. RNAP II ChIP

served as a positive control, showing that actinomycin D effectively blocked the

 

recruitment of RNAP II to the viral genome (Fig. 4.3D). These results suggest that

157

transcription by RNAP 11 per se may contribute to the depletion of histones from the
HSV-1 genome during lytic infection.

Interestingly, actinomycin D treatment also decreased the VP16 occupancy on IE
gene promoters, particularly at 4 hpi (Fig. 4.3E). Although this could be due to the fact
that actinomycin D is a DNA-intercalating drug and as such might prevent the binding of
VP16 to DNA, it is also possible that the increase in histone occupancy on IE gene

promoters diminishes the binding of VP16 to these regions.

158

 

 

Figure 4.3: Inhibition of transcription by actinomycin D increases the core histone
occupancy on the HSV-1 genome. HeLa cells were pretreated with 1 ug/ml of
actinomycin D or mock treated (DMSO) for 1 h and then were infected with the KOS
strain of HSV-1 at an MOI of 1 pfu/cell in the presence of 1 ug/ml of actinomycin-D.
Chromatin immunoprecipitation was performed to detect the occupancy of histones H2A
(A), HZB (B), H3 (C), RNA polymerase II (D), and VP16 (E) on the ICP27, ICPO, tk, and
gC promoters (pro) and on the ICP27 ORF at 2, 4, and 6 h post-infection. The data were
analyzed as explained in Materials and Methods. The graphs show the results of a
representative experiment; similar results were observed in independent replicates.

159

 

 

A. Histone H2A ChiP

 

 

4" iCP21pro H
+ lCP27pro (+)
4- iCPOpro H
+ "3pr (+)
-°- ICPZTORF H
+ ICP27ORF (+)

 

 

a
in
2
U-
i
E
o
a
E
a
a.
E

 

hours post infection
Histone HZB ChIP

 

 

4" iCP27pro H
+ iCPZTpro (+)
-A- iCPOpro H
+ ICPODI'O (+)
-°- iCP270RF (-)
+ ICPZTORF (+)

 

 

input ratio (virailiFN-Bp) P

 

hours post infection

Histone H3 ChIP

input ratio (viralIiFN-Bp) _0

hours post infection

iCP27 pro H
(+)
i-)

it)
i-)

 

 

P

RNAP ll ChIP

 
  
 

 

'0' iCP27pro H
+ iCPZTpro (+)
4- iCPOpro H
+ iCPOpro (+)
-0- iCP270RF H
+ iCP210RF (+)

 

 

  

2
hours post infection

4 6

160

Input ratio (vlraillFN-Bp)

Input ratio (viraiIIFN-Bp)

input ratio (viral/lFN-Bp)

%input (lP-noab)

0.4

 

 

-- tkpro (-i
-- tkpro (+)
-°- ecpro H
+ 11ch (+)

 

 

2 4
hours post infection

    

9 .0
A0

%

_.-°-

- . a
2 4 6
hours post infection

 

0.8
0.6
0.4
0.2 _ _ - S = 1 a
0.0
2 4 6
hours post infection
VP16 ChIP
0.3 ’ A ~ ~
0.2 , r ' “
r

G
.
.L

2 4
hours post infection

 

 

-- mm H
+ tkpro (+)
-°- 009m H
+ 13ch (+)

 

 

 

-- mm H
+ tkpro (+)
-°- 909m H
+ ecpro (+)

 

 

 

-- tkpro H
+ tkpro (+)
-°- ccpro H
+ some (+)

 

 

 

 

 

 

IE proteins are not required for inhibition of histone deposition on IE genes
during lytic infection.

During RPS infections or in the presence of actinomycin D, histone occupancy
increased not only at IE genes but also at DE and L gene promoters, where VP16 does
not bind directly. Given that IE proteins play important roles in transcriptional regulation
of DE and L genes, we considered whether IE proteins also contribute to the depletion of
histones from these genes. The hypothesis that histones are removed from DE and L
genes by IE proteins leads to the prediction that inhibition of IE protein synthesis would
increase the occupancy of histones on these promoters but would not affect IE genes,
which are actively transcribed by VP16 and RNAP II in the absence of IE proteins.
Therefore, we tested whether inhibition of IE protein expression by cycloheximide
affected histone deposition on the viral genome during lytic infection.

To this end, HeLa cells were infected with KOS in the presence of cycloheximide
to block de novo viral protein synthesis. At 2 and 4 hpi, infections were stopped and ChIP
assays were performed to analyze histone H3 and RN AP 11 occupancy on the HSV-1
genome. As expected, histone H3 occupancy on the ICPO promoter and the ICPO and
ICP4 ORFS was minimally affected by inhibition of IE protein expression at 2 hpi (Fig.
4.4A). H3 occupancy increased modestly on the ICP27 promoter and ICP27 ORF, as
well as on the tk and gC promoters (Fig. 4.4A). Similarly, at 4 hpi histone deposition did
not change substantially on the ICPO promoter, ICPO ORF, and ICP4 ORF (Fig. 4.43),
yet increased dramatically on the ICP27 promoter, ICP27 ORF, and the tk and gC
promoters (Fig. 4.43). These results suggest that IE proteins are not involved in depleting

the histones fiom IE promoters and ORFS, with the exception of ICP27.

161

Interestingly, the increase in histone H3 occupancy on the ICP27 promoter, ICP27
ORF, and tk and gC promoters corresponded to a significant decrease in RN AP 11
occupancy (Fig. 4.4C). As expected, RNAP II occupancy on other IE genes either did not
change or increased in the presence of cycloheximide (Fig. 4.4C). These results together
suggest that during early times in lytic infection, VP16-mediated transcription keeps the
IE genes relatively free of histones, and at later stages IE proteins are involved in the

removal of histones from other regions of the viral genome.

162

 

 

A. Histone H3 ChIP-thi

[3 HOW!
.2
E 0.2
30.1
.5
0.0

lCPOp iCPO ICP4 ICP27p ICP27 tkp ng
ORF OR ORF

B. Histone H3 ChIP-4hpi
El HCHX
I (*)CHX

.°
N

input ratio
9999999
9 ‘- NU # (I G

ICPOp ICPO ICP4 ICP27p ICP27 tip 909
ORF OR ORF

C. RNAP || ChIP-4hpi

El i-iCHX
.0 1.0 I men):
'5 1.2
‘5 0.0
n.
.E 0.4
0.0

ICPOp ICPO ICP4 ICP27): lCP27 tip 904)
ORF ORF ORF

Figure 4.4: Changes in histone occupancy on the HSV-l genome upon inhibition of IE
protein synthesis by cycloheximide. HeLa cells were pretreated with 60 ug/ml
cycloheximide for 2 h and then were infected with HSV-1 KOS at an MOI of l pfu/cell
in the presence of cycloheximide. (A) At 2 h post-infection, chromatin was crosslinked
and a ChIP assay probing histone H3 on the ICPO promoter (ICPOp), ICP27 promoter
(ICP27p), ICPO ORF, ICP4 ORF, ICP27 ORF, tk promoter (tkp), and gC promoter (ng)
was performed as indicated before. (B,C) At 4 h post-infection, chromatin was
crosslinked and a ChIP assay probing histone H3 (B) and RNAP II (C) on the indicated
viral gene fragments was performed as indicated above. The data shown represent the
average of two independent experiments; the error bars indicate the range between the
averages of these experiments.

I63

Histones are neither modified nor removed from the RPS genome upon
superinfection with HS V-2.

We designed an experiment to test whether VP16 can induce the removal of
histones that are already deposited on the viral genome. The VP16 proteins of HSV-1 and
HSV-2 share 93% sequence similarity (9). Moreover, HSV-2 superinfection can
reactivate quiescent HSV-l infections in vitro (8), and the VP16 gene from HSV-2 can
function effectively when recombined into the HSV-1 genome (52). These observations
indicate that the HSV-2 VP16 protein is capable of activating transcription from the
HSV-l genome. Given that RPS genomes associate with higher levels of histones at later
stages of infection (Fig. 4.2), we asked whether VP16 provided in trans by HSV-2
superinfection can overcome the nucleosomal barrier and induce IE gene expression from
the quiescent and histone-laden RPS genome.

We first tested whether VP16 fi'om HSV-2 superinfection could stimulate IE gene
expression from RPS genomes. HeLa cells were infected with RPS at an MOI of 0.001
pfu/cell. At 6 hpi, when the RPS viral genomes are highly associated with histones (see
Fig. 4.2), the cells were superinfected with HSV-2 at a range of multiplicities. RPS IE
gene expression was analyzed after 2 h of HSV-2 superinfection (Fig. 4.6A). As
expected, transcription of the RPS IE genes ICP4 and ICP27 was induced significantly
upon HSV-2 superinfection in a dose-responsive manner with respect to HSV-2 MOI
(Fig. 4.6B). RPS IE gene expression was also stimulated when the HSV-2 superinfections
were performed in the presence of cycloheximide (Fig. 4.5), indicating that the induction
of IE gene expression is largely attributable to VP16 but does not depend on de novo

synthesis of HSV-2 IE proteins such as ICPO.These results together indicate that VP16 of

164

HSV-2 can efficiently stimulate IE gene expression from viral genomes that are
predominantly nucleosomal.

We next tested whether VP 1 6 can induce changes in the chromatin structure of
RPS genomes upon superinfection with HSV-2. We hypothesized that if VP16 is
involved in the modification and removal of histones, then active transcription marks
such as H3 K9/Kl4ac or H3K4me3 would increase and the histone H3 occupancy on the
RPS genomes would decrease upon HSV-2 superinfection.

To address these questions, we performed ChIP assays in cells infected with RPS
and superinfected with HSV-2. HeLa cells were infected with RPS at an MOI of 0.001
pfu/cell. At 6 hpi, these cells were superinfected with HSV-2 at 10 pfu/cell, which
robustly induced RPS IE gene expression (Fig. 4.6B). Infections were stopped by
formaldehyde cross-linking after 2 h of HSV-2 superinfection, and the occupancy of
RNAP II, histone H3, H3K9/Kl4ac, and H3K4me3 on RPS IE promoters was analyzed
by ChIP.

As expected, RNAP II occupancy increased significantly at IE gene promoters
and open reading frames upon superinfection with HSV-2 (Fig. 4.6C). Even so, RNAP II
occupancy on the IE promoters was still 10—20 % of that observed on the cellular control
U3 snRNA promoter, indicating that the relative rate of transcription was lower on IE
genes than on the U3 snRNA gene. This result may suggest that only some, but not all,
RPS genomes are transcriptionally active upon HSV-2 superinfection. Interestingly, this
increase in RNAP II occupancy did not correlate with a significant decrease (p > 0.05, by
Student’s t-test) in histone H3 occupancy on IE promoters and ORFS (Fig. 4.6D),

suggesting that neither the VP16 AD nor RNAP II are sufficient to deplete histones from

165

the viral genome. No change in the occupancy of H3K9/K14ac (Fig. 4.6E) or H3K4me3
(Fig. 4.6F) on RPS IE genes was observed after superinfection with HSV-2, indicating
that VP16 does not induce major changes in covalent histone modifications on the viral
genome. These results collectively suggest that VP16 and RNAP II are not sufficient to
alter the chromatin structure on IE promoters. Perhaps more importantly, neither histone
removal nor covalent modification of histones seems to be a prerequisite for VP16-

mediated transcription from nucleosomal viral genomes.

166

 

 

D ICP4 I ICP27

 

C1000-
.9
8100-
“.3
8 10.
Q)
E 1-
n
a.» MI
1» r»
q?” ,o‘d‘ to“ 9“
a .2?‘ X‘+
4&3 Q‘” ebo‘f
an ex

Figure 4.5: HSV-1 IE gene expression is induced by HSV-2 superinfection independent
of de novo protein synthesis. HeLa cells were infected with the RPS strain of HSV-1 at an
MOI of 0.001 pfu/cell. At 5 hpi, cells were either treated with 60 ug/ml cycloheximide or
mock-treated for 1 hr. At 6 hpi, HSV-2 superinfection was started at an MOI of 10
pfu/cell either in the presence or absence of 60 ug/ml cycloheximide. Parallel samples
were mock-infected in the presence or absence of cycloheximide. At 2 h after HSV-2
superinfection, RNA was isolated and expression of HSV-1 IE gene ICP4 and ICP27 was
analyzed by Q-RT-PCR. The data shown are from a representative experiment
performed in biological triplicate. Error bars represent the standard deviation among
these samples.

167

Figure 4.6: HSV-2 superinfection does not cause major changes in histone occupancy or
covalent histone modifications on the RPS genome. (A) Summary of the assays
performed. (B) HeLa cells were infected with RPS at an MOI of 0.001 pfu/cell and at 6
hpi were superinfected with HSV-2 at MOIs of 0.1, 1, or 10 pfu/cell. At 2 h after HSV-2
superinfection, ICP4 and ICP27 expression was analyzed by Q-RT-PCR. (C-F) HeLa
cells were infected with RPS at an MOI of 0.001 pfu/cell and at 6 hpi were superinfected
with HSV-2 at an MOI of 10 pfu/cell. At 2 h after HSV-2 superinfection, ChIP was
performed assaying the presence of RNAP II (C), histone H3 (D), H3K9/Kl4ac (E), and
H3K4me3 (F) on the ICPO promoter, ICP27 promoter, ICP4 ORF, and ICP27 ORF. The
data shown represent the average of three independent experiments, where “% input”
values fi'om viral genes are normalized against the U3 snRN A promoter as indicated
before. Error bars represent the standard deviation between these experiments.

168

HeLa

RP5 infection
1 6 hr

HSV-2 infection
( 2 hr

Q-RT-PCR or ChIP

C
RNAP II ChIP
A 0.30
o. n RP5
‘3 015 I RP6+HSV-2
=
g 0.20
30 15
.2 -
E 0.10
a 0.05
C
" 0.00
ICPOp iCP27p ICP27 iCP4
ORF ORF
E
AcH3 ChIP
n RP5
a 0'04 I RP5+H8V~2
3
a 0.03
E
2.
o 0.02
.a
E
S 0.01
a.
.E
0.00
ICPOp ICPZ‘Ip ICP27 ICP4
ORF om:

169

relative expression

U

input ratio (viral/U3p)

11

input ratio (viral/U3p)

 

10000 +iCP4
-l-ICP27
1000
100
1 I l l 1
0.1 1 10
HSV-2 MOI
Histone H3 ChIP
2.0
El RP5
1.5 I RP5+HSV-2
1.0
0.5
0.0
iCPOp iCP27p iCP27 iCP4
ORF ORF
MeH3 ChIP
010 U RP5
I RP6+HSV-2
0.15
0.10
0.05
0.00
ICPOp ICP27p iCP27 iCP4
ORF ORF

Histones and RNAP II partially co—occupy the RP5 genome upon HS V-2
superinfection.

Although the results in Fig. 4.2 indicated a significant increase in histone
occupancy on the RP5 genome at 6 hpi, we cannot be certain that all RP5 viral genomes
are occupied by histones (i.e., the input ratio may be somewhat less than 1). In addition, it
is likely that not all RPS genomes were reactivated in response to HSV-2 superinfection,
as indicated by the lower occupancy of RNAP II on IE genes than on the U3 snRN A
promoter (Fig. 4.6C). These observations raise the possibility that VP16 and RNAP II
may preferentially associate with viral genomes that remain non-nucleosomal. This could
explain why no changes in histone occupancy or covalent histone modifications were
evident on the RPS genomes after HSV-2 superinfection (Fig. 4.6). Therefore, we
performed sequential ChIP assays to probe this issue.

Since histones H2B and H3 are expected to co-occupy a given cellular gene at
high levels, as control assays for histone-RNAP II sequential ChIPs we first addressed
whether histone H2B and H3 co-occupy cellular and viral genes. HeLa cells were
infected with RPS and superinfected with HSV-2 as in previous experiments. The cross-
linked chromatin from infected cells was first subjected to immunoprecipitation using a
histone H2B antibody, followed by a second immunoprecipitation with a histone H3
antibody (Fig. 4.7A). As expected, histones H2B and H3 co-occupied both the IFN-B and
the U3 snRNA promoters (Fig. 4.7A). We noted that the level of histone co-occupancy
on the U3 snRN A promoter was less than that on the IFN-B promoter, which might
indicate active transcription on the former promoter. Histones H2B and H3 also co-

occupied the viral IE genes at levels similar to those on cellular promoters (Fig. 4.7A).

170

 

Similar results were observed when the first IP was performed using H3 antibodies
followed by a second immunoprecipitation by H2B antibodies (Fig. 4.7B). These results
collectively indicate the feasibility of sequential ChIP assays in this experimental context.
Interestingly, the association of H3 and H2B at viral genes was comparable to that at the
cellular genes, indicating co-association of these histones on viral DNA. This
observation is consistent with the idea that entire nucleosomes form on the viral genome.
Co-occupancies of histone H3 and RNAP 11 (Fig. 4.7C) and of histone H2B and
RNAP 11 (Fig. 4.7D) were analyzed similarly by sequential ChIP in RP5-infected and
HSV-2-superinfected cells. The cross-linked chromatin was first subjected to
immunoprecipitation with RNAP II antibodies, followed by a second
immunoprecipitation histone H3 or histone H2B antibodies using chromatin material
obtained from the first IP. We used the U3 snRN A promoter as a positive control. As
expected, RNAP II and histone H3 (Fig. 4.7C) or RNAP II and histone H2B (Fig. 4.7D)
co-occupied the U3 snRNA promoter. Interestingly, RNAP II and histone H3 co-
occupied the RPS ICPO promoter and ICP27 ORF at levels similar to that of the U3
promoter, yet the co-occupancy was significantly lower on the ICP27 promoter (Fig.
4.7C). RNAP II and histone H2B also co-occupied IE genes (Fig. 4.7D), yet the degree of
co-occupancy was significantly lower than on the U3 snRNA promoter for some IE genes
such as the ICP27 promoter and ICP27 ORF. These results suggest that although RNAP
11 does not preferentially associate with “histone-free” RP5 genomes upon HSV-2
superinfection, VP16 and RNAP II-mediated transcription may lead to the disruption of
nucleosome structure. One possible mechanism is by evicting a histone H2A-H2B dimer

from the nucleosome core particle, as observed in other systems (19, 33).

171

In order to gain more confidence in these results, we analyzed RN AP 11 and
histone co-occupancy on genes that associate with either RN AP 11 or histones, but not
both. The promoter region of the transcriptionally inactive IFN-B gene associates with
histones but not RNAP II. As expected, neither of the histones co-occupied the IFN-B
promoter with RNAP II, as indicated by the absence of IFN-B-specific amplification in
PCR samples parallel to those presented in Figures 4.7C and 4.7D (data not shown). As a
second negative control, we analyzed the co-occupancy of RN AP 11 and histones on
genes that are associated with RNAP II but low levels of histones. To this end, we
repeated the sequential ChIP assays in KOS-infected cells and asked whether histones
and RN AP 11 co-occupy the viral IE genes. Given that histone levels are low on the KOS
genomes (Fig. 4.2, 4.3), we expected that the viral genomes that are associated with
RNAP 11 would be low in histones also. As expected, histone H3 was underrepresented
on all viral IE promoters that are associated with RNAP 11 (Fig. 4.7E). Interestingly,
histone H3 was depleted from the ICP27 ORF to a lesser extent than from IE promoters.
Similarly, the co-occupancy of histone H2B and RNAP II on IE promoters and the ICP27
ORF was significantly lower than on the U3 snRNA promoter (Fig. 4.7F). These results
together reinforce our conclusions from sequential ChIP assays performed in RPS-

infected and HSV-2-superinfected cells.

172

 

 

Figure 4.7: Histone and RN AP II co-occupancy on RP5 IE genes upon HSV-2
superinfection. HeLa cells were infected with RPS at an MOI of 0.001 pfu/cell and at 6
hpi were superinfected with HSV-2 at an MOI of 10 pfu/cell. At 2 h after HSV-2
superinfection, seq-ChIP was performed assaying the co-occupancy of histone H3,
histone H2B, and RNAP II on the U3 snRN A promoter, IFN-B promoter, ICPO promoter,
ICP27 promoter, and ICP27 ORF. (A) Histone H2B (first immunoprecipitation) and H3
(second immunoprecipitation) co-occupancy. (B) Histone H3 (first immunoprecipitation)
and H2B (second immunoprecipitation) co-occupancy. (C) RNAP 11 (first
immunoprecipitation) and H3 (second immunoprecipitation) co-occupancy. (D) RNAP 11
(first immunoprecipitation) and H2B (second immunoprecipitation) co-occupancy. (E)
HeLa cells were infected with KOS at an MOI of l pfu/cell and seq-ChIP was performed
at 2 hpi as in (A-D). RNAP 11 (first immunoprecipitation) and H3 (second
immunoprecipitation) co-occupancy. (F) Seq-ChIP was performed in HeLa cells infected
as in (E). RNAP II (first immunoprecipitation) and H2B (second immunoprecipitation)
co-occupancy. The data shown in (A, C, D) represent the averages of three independent
experiments, with error bars representing the standard deviation between these
experiments. The data in (B) represents the average of two independent experiments, with
error bars representing the range between these experiments. The data in (E, F) represents
three independent IPs done in parallel, where the error bars represent the standard
deviation. Samples with mean values that vary significantly (p S 0.01 or else 0.01 S p _<_
0.05 by paired student’s t test) from the U3 snRN A promoters in (C-F) are indicated by
C”) and (*), respectively.

173

>

% input (IP-noab)

O

96 input (IP-noab)

I11

56 input (iP-noab)
o 8

RP5 + HSV-2
H23 (1st), H3 (2nd)

59 99 g9 «9 Q3
° <<“ \‘3 ‘6’” 94°

RP5 + HSV-2
Poi2 (1st), H3 (2nd)

174

% input (lP-noab)

U

%inputflP-noab)
O -h N 0) -L

‘n

% input (iP-noab)

4.3
wt»

o-r-m

RP5 + Hsv-2
H3 (1st), H23 (2nd)

R95 + HSV-2
90:2 (1st), H23 (2nd)

Q Q «Q Q
0‘!» QB t), 0(-
‘9 £3 ‘30

5. DISCUSSION

An increasing amount of evidence indicates the important role of chromatin
regulation during the lytic and latent stages of HSV-1 infection. Recent studies have
shown that histone H3 associates with the HSV-l genome at lower levels than with
cellular genes during lytic infection (20, 21, 26, 35, 50). Active transcription marks such
as H3K9/Kl4ac and H3K4me3 are also present on the viral genome during lytic infection
(20, 21 , 26). In contrast, during latency the viral genome is chromatinized to a larger
extent, and the histones associated with the viral genome carry inactive chromatin marks
with the exception of the actively transcribed LAT gene (10, 32, 66). In addition, during
reactivation from latency and quiescence, acetylated histones associate with IE gene
promoters (8, 49). These results all indicate that chromatin is dynamically regulated
during different stages of HSV-1 infection and that the transcriptional status of the viral
genome correlates with the type of covalently modified histones associated with it. On
the other hand, the mechanism of how the viral genome stays predominantly non-
nucleosomal during lytic infection has not been studied in detail.

In this study we addressed potential mechanisms to account for the dearth of
histones on the viral genome during lytic infection. We have previously analyzed the role
of transcriptional coactivators that are recruited by VP16 in this process and showed that
disrupting the expression of a number of coactivators did not reduce IE gene expression
during lytic infection, suggesting that these coactivators are not likely involved in
modulating histone occupancy on IE genes (3 6). We thus turned our attention to other
possible mechanisms and analyzed whether the VP16 AD, transcription by RNAP II per

se, or IE proteins affect histone levels on the viral genome.

175

 

 

Our results show that the activation domain of VP16 contributes to low histone
levels on the HSV-l genome, as the absence of the VP16 AD resulted in a dramatic
increase in the occupancy of all core histones on the RPS genome (see Fig. 4.2). An
indirect mechanism whereby the VP16 AD might mediate the removal of histones from
the viral genome is simply by recruitment of host RNAP II machinery, as it has been
shown that histones are underrepresented on actively transcribed genes (4, 19, 44, 45,
55). In support of this notion, histone occupancy dramatically increased throughout the
viral genome in the presence of the RNAP II inhibitor actinomycin D (see Fig. 4.3). This
result indicates that transcription per se reduces histone occupancy on viral genes. This
result might be further interpreted to mean that the VP 1 6 AD and whatever coactivators it
recruits are not sufficient to maintain low histone levels on the viral genome without
active transcription by RNAP II. This interpretation is complicated by the observation
that VP16 occupancy on IE promoters at later times post-infection was also reduced by
actinomycin D. The loss of VP16 binding might be a consequence of increased histone
occupancy on these promoters, blocking access by the VP16-induced protein complex.
An alternative possibility is that actinomycin D directly prevents the binding of VP16 and
its partners to IE promoters, which may in turn lead to an increase in histone occupancy.

IE proteins themselves may also affect histone levels on the viral genome, as
indicated by the increase in histone occupancy on DE and L gene promoters in the
absence of IE protein expression (see Fig. 4.4). As expected, inhibition of IE protein
expression did not affect histone occupancy on most IE genes; the exception was the
ICP27 gene, both the promoter and ORF. We currently do not have a clear explanation of

why the ICP27 gene is regulated differently than other IE genes. An important point to

176

 

note is that RN AP 11 occupancy on the ICP27 gene was reduced substantially, much like
on the DE and L promoters in the presence of cycloheximide, in contrast to the enhanced
RNAP II recruitment to other IE genes (see Fig. 4.4C). Therefore, it is likely that de novo
IE protein synthesis is necessary for the maximal transcription of ICP27, but not of the
other IE genes.

One mechanism by which IE proteins might mediate histone depletion is the
recruitment of RNAP II and the induction of active transcription. Alternatively, IE
proteins might be directly involved in regulating histone deposition on the viral genome.
For instance, ICPO was shown to disintegrate the REST/CoREST/HDAC repressor
complex (14, 15) and thus was suggested to prevent the formation of inactive chromatin
on the HSV-1 genome. Cliffe and Knipe (6) have recently shown that this function of
ICPO may be relevant during lytic infection, as in the absence of ICPO, histone H3
occupancy increased and the fraction of acetylated histones on the viral genome
decreased significantly. Whether the REST/CoREST/HDAC complex is the only target
of ICPO and whether the changes in chromatin on the viral genome in the absence of
ICPO are directly mediated by REST/CoREST/HDAC still need to be determined.

Although our results indicate that VP16 and RN AP 11 contribute to the dearth of
histones on IE gene promoters and ORFS during lytic infection, reduced histone levels are
not required for IE gene transcription. Histones were neither modified on nor depleted
fi'om RPS genomes upon superinfection with HSV-2 (see Fig. 4.6), in spite of the
induction of RPS IE gene expression by HSV-2 (see Fig. 4.6B). These results suggest that
the presence of histones may not interfere with the ability of VP16 and RNAP II to

initiate transcription from the viral genome.

177

 

A potential complication in these assays is the possibility that not all viral
genomes are activated by HSV-2 superinfection, as indicated by the low levels of RN AP
II occupancy on IE genes (Fig. 4.6C). This might explain why no change in histone
occupancy or modifications was observed on the RPS genome after HSV-2
superinfection. We think that similar problems might be faced when studying reactivation
from latency in vivo, where only a fi'action of latent viral genomes might get reactivated.

Given the possibility that not all RPS genomes become transcriptionally active
upon HSV-2 infection and that some RPS genomes may escape histone deposition, we
hypothesized that the RPS genomes that are activated by HSV-2 superinfection might
actually be the ones that escape nucleosome deposition. However, sequential ChIP assays
showed that RNAP II has no clear preference for histone-free RPS genomes (see Fig.
4.7C, D). We conclude that removal of histones from viral templates is not a prerequisite
for transcriptional activation by VP16. Interestingly, histone H3 and H2B were
significantly underrepresented in some RNAP II-associated IE genes (Fig. 4.7C, D),
suggesting that nucleosomes might be partially disrupted at actively transcribed IE genes.
A similar phenomenon was suggested for transcriptionally active eukaryotic genes (19,
33)(30,38,S8)

Another important question addressed in this study is the nature of chromatin on
the viral genome during lytic infection. We suggest that the presence of all core histones
on the viral genome during KOS and RP5 infections is indicative of formation of
canonical nucleosomes on the viral genome. This conclusion is supported by our findings
in sequential ChIP assays, which indicated that histones H2B and H3 co-occupied the

RPS genomes in HSV-2 superinfected cells (see Fig. 4.7A, B).

178

 

A crucial question is whether the low histone occupancy on the HSV-l genome
during lytic infection matters for transcription. A related issue is whether the dearth of
histones on the viral genome during lytic infection is due to prevention of histone
deposition or effective removal of histones that are deposited. In contrast to a recent
report (6), we have noted that even at very early times in infection (30 min to l h), the
amount of histones on the viral genome is much lower than on cellular genes (data not
shown), suggesting that histones may not be deposited at all on most viral genomes
during lytic infection.

One possible model is that efficient transcription, which starts soon after the viral
genome is released into the nucleus, might block histone deposition on the HSV-l
genome. If transcription does not start efficiently, as in the case of RP5 infections or in
the presence of actinomycin D, then the histones are deposited on the viral genomes
rather slowly as indicated by the time-course assays in Figures 4.2 and 4.3. Therefore, we
propose that histones are prevented from being deposited on the viral genome, rather than
being deposited first and then actively removed. If this is the case, then the small amount
of histones on the viral genome may not matter for viral transcription during lytic
infection. In this scenario, the small amount of histones might represent randomly
positioned histones on the viral genome. A second alternative is that during wild-type
lytic infection, a small fraction of viral genomes are silenced by deposition of
nucleosomes. In either case, histones may not matter for the outcome of lytic infection, as
a large fraction of actively transcribed viral genomes stay non-nucleosomal.

Given that histones are not present in the HSV-1 nucleocapsid (7, SO, 53) and are

underrepresented on the HSV-l genome during lytic infection, it is tempting to speculate

179

 

that histone chaperones and assembly factors may be involved in regulating the viral
chromatin structure. Some of the candidate chaperones include Spt6 and FACT, which
associate with RNAP II and enable its elongation on nucleosomal templates (2, 25).
Interestingly, the histone chaperone HIRA, which is involved in replication-independent
histone deposition (1 8, 57), was shown to be present in PML bodies in senescent cells
(69) and as such is likely to be involved in chromatin assembly on the viral genome. A
recent study suggested that disruption of HIRA expression reduces both the association of
histone H3.3 with the HSV-l genome and the viral gene expression at later stages of
infection, concomitant with a reduction in viral replication (54). On the other hand, it is
not clear whether HIRA contributes to viral gene expression at early times in infection.

Nucleosome assembly on the viral genome at early times may be blocked by
VP22, a tegument protein that interacts with TAF-I, a homologous protein to histone
chaperone NAP-l (64). However, our results (see Figures 4.2 and 4.3) suggest that VP22
delivered by the incoming virion is not sufficient for histone depletion, because the levels
of VP22 are presumably unaffected by the absence of the VP16 AD or the presence of
actinomycin D. Given these findings, it will be important to address whether histone
chaperones are modulated in a way that leads to low levels of histones on the HSV-l
genome during different stages of infection.

Overall, we show that during lytic infection of HSV-1, histones are found at low
levels on the viral genome; the cause of such levels may involve active transcription by
RNAP II, as well asVPl6 and IE proteins. Future studies should address the details of the
underlying molecular mechanism in terms of histone deposition on or histone removal

from the HSV-1 genome. In addition, our results suggest that IE proteins also contribute

180

 

to keeping other regions of the viral genome free of nucleosomes at later stages of lytic
infection. Therefore, it will be crucial to define the cellular factors involved in depositing
the histones on the viral genome. Lastly, it will be interesting to see whether and how
chromatin assembly factors are regulated during establishment of and reactivation from

latency of HSV-1.

6. ACKNOWLEDGEMENTS
This research was supported by the Van Andel Research Institute and by NIH grant
AIO64634. SK was supported by a predoctoral fellowship from the American Heart
Association. We thank Martha Roemer for initiating the chromatin immunoprecipitation
analyses of core histones. We also thank David Nadziejka for assistance in preparing the

manuscript and members of the Triezenberg lab for their comments on this work.

181

10.

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187

Chapter 5
SYNTHESIS AND FUTURE DIRECTIONS

This dissertation contributes significantly to our understanding of how the HSV-1
genome stays predominantly fi'ee of nucleosomes during lytic infection. Although the
molecular details remain to be elucidated, histones are likely prevented from being
deposited on the HSV-1 genome during lytic infection by a complex mechanism that
involves VP16, RNAP II and IE proteins (Chapter 4), rather than being deposited on the
viral genome first and then being removed from it. This mechanism explains why the
transcriptional coactivators that are recruited by VP16 are not required for IE gene
expression during lytic infection (Chapter 2). A number of issues that arise from these
observations are discussed below.

1. Potential mechanisms leading to the lack of histones on the HSV-1 genome
during lytic infection

Although VP16, RNAP II and IE proteins contribute to keeping the viral genome
relatively free of histones during lytic infection, the mechanistic details of this process
remain unknown. In this section I will discuss the potential molecular mechanisms of
how these factors may lead to the dearth of histones on the viral genome during lytic
infection.

Although our initial studies using KOS and RPS viruses indicated that VP16 AD
contributes to keeping IE, DE and L genes depleted of histones (17), it is now clear that
the role of VP16 in this process is restricted mainly to IE genes, indicated by the increase
in histone occupancy on DE and L, but not IE genes, in the presence of cycloheximide.

One way that VP16 may block histone deposition on the viral genome is by recruitment

188

of RN AP II machinery. Alternatively, VP16 may recruit other factors, which in turn
block histone deposition on or induce rapid removal of histones fiom the viral genome.
Some of these factors, such as transcriptional coactivators and proteins associated with
the RNAP II machinery, that interact with VP16 AD have been identified in vitro (see
Chapter 1). However, some of these interactions may not be relevant during HSV-1 lytic
infection as evidenced by the lack of change in viral gene expression in infected cells
when the expression of transcriptional coactivators that interact with VP16 AD in vitro is
disrupted (Chapter 2). Interestingly, a yeast-two-hybrid system using Ga14-VP16 AD
fusion as bait and a human cDNA library as prey identified a number of interaction
partners for VP16 AD, such as proteins without any known functions in addition to
proteins associated with RNAP II machinery (l 8). On the other hand, in this same study,
known in vitro interaction partners of VP16 AD were not identified, suggesting that
VP16 may interact with a different set of proteins in vivo. As such, it will be important to
identify the in vivo interaction partners of VP16 AD during genuine HSV-1 lytic
infection, which might clarify the mechanistic details of how VP16 activates IE gene
expression and how it regulates the structure of chromatin on IE genes. Identifying the in
vivo interaction partners of VP16 during infection, however, is a challenging task given
the need for large volumes of cells and viruses to isolate VP16 containing protein
complexes by mass-spectrometry applications.

Although hypothetical, another mechanism that VP 1 6 may prevent histone
deposition is by directing the viral genomes to microdomains in the nucleus that may be
rich in RNAP II and other transcription factors but poor in histones. This model would

explain why DE and L genes that are not transcribed at early times in infection still

189

remain relatively free of histones. In this scenario, RNAP II recruitment might still be the
rate-limiting step, which might be overcome by transcriptional activators, such as VP16
at early times and ICP4 at later times in infection.

The idea of the presence of microdomains surrounding the viral genome is not
unprecedented; upon the entry of the HSV-l genome to the nucleus, PML bodies form
around the viral genome as part of the innate immune response. Although the association
of the viral genomes with PML bodies is thought to result in transcriptional silencing of
the viral genome (10, 28), not all components of PML bodies may be inhibitory for viral
gene expression. For instance, transcriptional coactivators such as p300 and CBP HATS
are also present in ND10 structures (23, 24, 26), although it is now clear that these HATS
are not necessary for viral gene expression (Chapter 2). Interestingly, when plasmid DNA
was artificially targeted to PML bodies, transcription was either inhibited or induced in a
promoter-dependent manner (2), supporting the idea that PML bodies comprise factors
that can both inhibit or activate transcription. Therefore, although PML bodies may be
formed around HSV-1 DNA as a host defense, HSV-1 may employ some of the PML-
associated factors for initiating viral transcription.

Whether VP16 has any role in the association of the HSV-1 DNA with PML
bodies is currently not known. Interestingly, the functional homologue of VP16 in human
cytomegalovirus, pp71, leads to dissociation of ATRX, a protein containing an ATPase
domain found in chromatin remodeling enzymes, from the PML bodies at early times in
infection (25). In this case, ATRX contributes to inhibition of IE gene expression and
hence its dissociation from PML bodies by pp7l potentiates IE gene expression (25).

Although VP16 does not share sequence homology with pp71, it is possible that VP16

190

also contributes to early changes in PML bodies to stimulate viral transcription. It will
therefore be important to address whether VP16 is involved in localization of the viral
genomes to PML bodies and whether it alters components of the PML bodies for optimal
viral gene expression during lytic infection. To this end, fluorescence in situ
hybridization and immunofluorescence assays in infected cells can be employed.

One important finding in line with the potential importance of nuclear architecture
for HSV-1 gene expression is the interaction between PML and special AT-rich sequence
binding protein 1 (SATBI) (22), which regulates higher-order chromatin structure and
transcription by attaching actively transcribed genes to the nuclear matrix and inducing
the formation of DNA loops that emanate from matrix associated regions. Therefore, it
would be interesting to address whether SATBl also colocalizes with the incoming DNA
and tethers it to the nuclear matrix, which might be important for the initiation of viral
gene expression. Another intriguing question then would be whether VP16 has a role in
this process. To address whether the incoming viral genomes are directed to the nuclear
matrix, one could employ the “in situ hybridization to nuclear halos” assay (5, 11). In this
assay, cells are perrneabilized by Triton X-100 treatment, and histones and soluble
nuclear proteins are extracted by high-salt treatment. 4,6-Diamidino-2-phenylindole
(DAPI) staining of these nuclei show that the regions that associate with nuclear matrix
form a DAPI-dense region, surrounded by a relatively faint DAPI staining (halo) which
represents the histone-rich heterochromatin regions. F lorescence in situ hybridization of
these nuclei reveals the association of a given DNA sequence with either heterochromatic
regions or the nuclear matrix. One could therefore use this assay in cells infected with

wild-type or the VP16 AD-deleted viruses to address whether the activation domain of

191

VP16 is involved in directing the viral genome to the nuclear matrix potentially rich in
transcription factors and poor in histones. One would then ask whether SATBl is
involved in mediating this attachment by performing similar assays in SATBl depleted
cells. Further assays would address whether VP16 and SATBl interact to direct the viral
genome to distinct regions in the nucleus.

In support of the role for nuclear architecture for regulation of viral gene
expression and histone deposition on the viral genome, a recent study indicated that
absence of lamin A, a major structural component of the nuclear lamina, leads to defects
in viral gene expression and replication as well as a significant increase in
heterochromatin formation on the viral genome (3 6). It will be important to elucidate the
underlying molecular mechanism and whether VP16 has any role in this process, i.e. by
localizing the incoming viral genomes in distinct regions in the nucleus by interacting
with lamin A.

The mechanism of how IE proteins prevent histone removal is currently not
substantiated, yet current evidence indicates an important role for ICPO in this process.
One way that ICPO may block histone deposition on the viral genome is by overcoming a
general silencing mechanism by disrupting the PML bodies (9). Interestingly, blocking
the disruption of PML bodies by overexpressing a structural component had no effect on
viral gene expression (24), indicating that disruption of PML bodies may not be
necessary for escaping fi'om silencing by PML. Another relevant function of ICPO is its
interaction with the REST/CoREST/HDACI repressor complex, which results in
dissociation of HDACl from the complex (12, 13). Whether this mechanism is relevant

to viral lytic infection is currently not known. It is interesting, however, that during lytic

192

infection by a mutant virus that lacks ICPO, the amount of histone H3 on the viral
genome increases significantly and the fraction of acetylated histones is reduced (6),
suggesting that ICPO may indeed be regulating histone occupancy and histone acetylation
on the viral genome during lytic infection. Interestingly the requirement for ICPO during
lytic infection is both dependent on the cell type and the multiplicity of infection,
suggesting that ICPO itself may not be necessary, at least in certain experimental settings,
to prevent chromatin formation on the viral genome. Finally, another mechanism that
ICPO may be contributing to histone depletion is simply by allowing active transcription
by RNAP II, as explained below.

Active transcription by RN AP 11 may also lead to partial or complete histone
depletion from gene promoters or transcribed regions (16, 21 , 40). In addition, recent in
vitro evidence indicated that passage of RNAP 11 leads to the elimination of an H2A-H2B
dimer from nucleosomes (20, 38). High-throughput genome-wide screens have also
shown that high rates of histone turnover within coding regions, but not promoters,
correlate with RNAP II density (8). One hypothesis posits that the density of RNAP II
molecules on a given template may itself block nucleosome formation (21). Although we
have shown in Chapter 4 that VP16, ICPO and transcription by RN AP 11 all contribute to
the dearth of histones on the viral genome, it is important to address more extensively
whether active transcription of the viral genome correlates with the level of histones
during lytic infection by ChIP assays. A number of mutant viruses that are debilitated in
expression of different temporal classes of viral genes can be employed to address
whether presence of RNAP II on a given viral gene correlates with histone depletion from

that gene. For instance, viruses that are deleted for DNA polymerase gene are debilitated

193

 

for L gene expression, but can express IE and DE genes. Therefore, during lytic infection
with these viruses, one would expect to observe an increase in histone occupancy on L
genes, but not on IE or DE genes.

Since a number of histone chaperones and chaperone-like proteins, such as FACT
and Spt6, associate with the elongating RNAP II, it is attractive to suggest that RNAP 11-
associated chaperones might regulate chromatin formation on the viral genome during
lytic infection. Although hypothetical, given that HSV-l infection dramatically changes
the phosphorylation of the C-terminal domain of RNAP II (32), it is possible that a
number of cellular chaperones (or other cellular proteins) might differentially interact
with this form of RNAP II and thus contribute to histone depletion from the viral
genome.
2. Transcriptional coactivators and HSV-1 infection

A surprising finding in our studies was that transcriptional coactivators which are
recruited by VP16 AD are not important for IE gene expression during lytic infection (see
Chapter 3). As it is likely that histones may be prevented from being deposited on the
viral genome rather than being deposited first and then removed from it, coactivators may
not be important for viral gene expression during lytic infection. This, however, does not
exclude the possibility that transcriptional coactivators are required in other contexts, i.e.
during reactivation from latency.

It is well established that the viral genome is nucleosomal during latent infections
(7) and the histones associated with the viral genome carry inactive transcription marks
(39). During reactivation fi'om latency, HSV-1 transitions from a nucleosomal state to a

non-nucleosomal one. Although there is conflicting evidence whether VP16 is required

194

 

during reactivation fi'om latency (14, 34), it may well be the case that recruitment of
coactivators by VP16 is important during this process. Interestingly, using the in vitro
“quiescent infection” model (see Chapter 2) we have shown that coactivators may not be
required during induction of IE gene expression by VP16 from heavily nucleosomal
templates, yet this system may not be representative of in vivo latent infections.
Addressing whether transcriptional coactivators are required during reactivation from
latency in vivo, however, is challenging given that the absence of most coactivators result
in embryonic lethality in mouse (3, 4, 29, 41, 42), a widely used animal model for HSV-1
latent infection.

As explained in Chapter 1 in detail, during latency in sensory neurons the HSV-1
genome is associated with histones that carry inactive transcription marks. Upon
reactivation from latency by stress stimuli, some of the viral genomes become
transcriptionally active leading to their replication and production of infectious virions
that are released by anterograde transport at the epithelial cells, the sites of lytic infection.
An important question regarding the state of viral chromatin during this process is at what
stage and by what mechanism the histones on the viral genome are depleted, such that no
histones are incorporated in the viral capsids produced in the sensory neurons. Although
there is evidence that reactivation from latency correlates with the appearance of active
transcription marks, such as H3K9/Kl4ac, on the transcriptionally active ICPO promoter
(1), it is currently not known whether histones are removed simultaneously with their
covalent modification or whether histone depletion is a later event following replication.
If the former model is the case, then one would expect blocking viral replication would

still lead to histone depletion on the actively transcribed viral genes. With regards to the

195

 

latter model, given that CAF -1, the replication-dependent nucleosome assembly factor,
interacts with the DNA polymerase clamp, proliferating cell nuclear antigen (PCNA)

(3 5), it would be informative to address whether CAF -1 also interacts with the HSV-l
processivity factor, UL42. The lack of such interaction would support the model that
upon replication of reactivated viral genomes, the newly replicated viral DNA would be
prevented from being nucleosomal and marked for packaging into the viral capsid. One
difficulty of testing these models is that only a small fraction of latent viral genomes are
reactivated, and as such the large fi'action of viral genomes that are still nucleosomal
upon reactivation stimuli may hinder observing the changes in histone levels on the
reactivated viral genomes.

Although a number of coactivators tested in this thesis (see Chapter 2) are not
required for the transcription of IE genes, a number of studies indicated a role for Setl
histone methyltranferase for optimal expression of viral genes and replication (19, 27).
Setl is recruited to IE gene promoters by interacting with HCF—l, a component of VP1 6-
induced complex. Interestingly, although the disruption of Setl by RNAi did not cause a
substantial decrease in IE gene expression at early times, it did cause a decrease in ICPO
and VP16 steady-state mRN A levels at late times in infection, concomitant with
replication (19). Hence, future studies will be necessary to address whether Setl is
required for IE gene expression. In addition, if Setl is also required for the expression of
other temporal classes of HSV-1 genes, it will also be important to address the
mechanism of how Setl would be recruited to other regions of the viral genome. On the
other hand, given that histones may not be deposited at all on the HSV-1 genome, we

think it is more likely that Setl is also not essential for viral gene expression.

196

3. Role of histone chaperones in HSV-l infections

The assembly of histones and histone variants into nucleosomes requires the
activities of a number of proteins and protein complexes (15, 33). CAF 1 and HIRA are
two such assembly factors that incorporate H3.l (canonical histone H3) and H3.3 into
nucleosomes in a replication-dependent and -independent manner, respectively. Another
histone chaperone, Asfl a, interacts with both CAFl and HIRA and is involved in both
replication-dependent and -independent histone assembly.

Given that during lytic infection histones are likely prevented from being
deposited on the viral genome, it is possible that chromatin assembly by histone
chaperones may be blocked by viral proteins. Interestingly, HIRA is a component of
PML bodies, which are disrupted by ICPO during lytic infection. Therefore, an attractive
model is that HIRA may be included in PML bodies to silence the incoming viral
genome, yet its activity or targeting may be blocked by viral factors such as ICPO, which
disrupts PML bodies (9). However, a recent report suggested that HIRA and histone
deposition on the viral genome may actually contribute to viral gene expression and
replication, rather than inhibiting it (30). On the other hand, in this same study, no
significant change in expression of certain viral genes was observed at early times in
infection. These results are also contradictory with the fact that most of the viral genomes
remain free of histones during lytic infection. In other words, if histone deposition on the
viral genome was necessary for viral gene expression, one would expect to observe more
histones on the actively transcribed viral genome. This, however, does not seem to be the
case as evidenced by our sequential-ChIP assays in KOS-infected cells (see Chapter 4),

where the actively transcribed IE genes are depleted of histones. Therefore, future studies

197

 

will need to address in more detail whether the presence of low amount of histones is
important for viral gene expression and whether the nucleosome assembly machinery in
host cells is altered in a way to prevent histone deposition on the viral genome. One way
to address the latter possibility is by checking whether HSV-1 infected cells are
debilitated in nucleosome assembly in vitro by DNA supercoiling assays (31, 37), which
allow testing both replication-dependent and —independent nucleosome assembly.
Alternatively, RN Ai and overexpression of histone chaperones and chromatin assembly
factors may be employed to address the necessity of these factors for viral gene
expression.
4. Concluding Remarks
Chromatin on the HSV-1 genome is clearly regulated differentially during lytic
and latent stages of infection. In this thesis, I tried to elucidate the mechanisms that lead
to dearth of histones on the viral genome during lytic infection and indicated a role for
VP16, IE proteins and RN AP II-mediated transcription in preventing histone deposition
on the viral genome. This mechanism points to an important concept, that is, regulation
of chromatin itself is not likely to determine whether the virus will initiate lytic infection
or stay latent. It is more likely that changes in the chromatin state of the viral genome is
a result of transcriptional activity mediated by VP16, IE proteins and RNAP II, and
possibly other transacting factors that still need to be determined. Therefore, identifying
these factors and the mechanisms of their action is crucial for our understanding of

regulation of chromatin during HSV-1 infections.

198

 

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