REVISED VERSION

Chromatinisation of herpesvirus genomes

Running head: Chromatinisation of herpesvirus genomes

Key words: herpesvirus, epigenetics, chromatin, nucleosome, histone, transcription

Christina Paulus, Alexandra Nitzsche and Michael Nevels*

Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany

*Corresponding author: M. Nevels, Institute for Medical Microbiology and Hygiene, University of

Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. E-mail:

michael.nevels@klinik.uni-regensburg.de

Abbreviations used

ASF1, anti-silencing function 1

ATRX, -thalassemia/mental retardation syndrome X-linked protein

BGLF4, EBV BamHI G left frame 4 protein

BHV-1, bovine herpesvirus type 1

BRG1, Brahma-related gene 1 protein

BRM, Brahma protein

BZLF1, EBV BamHI Z left frame 1 protein

CAF1, chromatin assembly factor 1

CBP, cAMP response element binding protein-binding protein

CENP-A, centromere protein A

ChIP, chromatin immunoprecipitation

CoREST, co-repressor to REST

Daxx, death domain-associated protein

EBER, EBV-encoded small RNA

EBNA, EBV nuclear antigen

GCN5, general control non-derepressible 5 protein

HAT, histone acetyltransferase

hCMV, human cytomegalovirus

HDAC, histone deacetylase

HMT, histone methyltransferase

ICP, infected cell protein

IE, immediate-early

K-bZIP, KSHV basic leucine zipper protein

K-RTA, KSHV replication and transcription activator

KSHV, Kaposi’s sarcoma-associated herpesvirus

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LANA, KSHV latency-associated nuclear antigen

LAT, HSV-1 latency-associated transcript

mCMV, murine cytomegalovirus

MHV68, murine -herpesvirus 68

ND10, nuclear domain 10

NuRD, nucleosome remodelling and deacetylase complex

oriP, EBV latent origin of DNA replication

PCAF, p300/CBP-associated factor

PML, promyelocytic leukemia

pp71, hCMV phosphoprotein 71

REST, repressor element 1-silencing transcription factor

Set1, SET domain containing family member 1

SNF5/Ini1, sucrose non-fermenting 5 homologue/integrase interactor 1

SUV39H1, suppressor of variegation 3-9 homologue 1

SV40, simian virus 40; SWI/SNF, switching/sucrose non-fermenting protein complex;

TAF1, template activating factor 1

UL, unique long; US, unique short

vIRF-1, viral interferon regulatory factor 1

VP, virion protein

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SUMMARY

The double-stranded DNA genomes of herpesviruses exist in at least three alternative global

chromatin states characterised by distinct nucleosome content. When encapsidated in virus

particles, the viral DNA is devoid of any nucleosomes. In contrast, within latently infected nuclei

herpesvirus genomes are believed to form regular nucleosomal structures resembling cellular

chromatin. Finally, during productive infection nuclear viral DNA appears to adopt a state of

intermediate chromatin formation with irregularly spaced nucleosomes. Nucleosome occupancy

coupled with posttranslational histone modifications and other epigenetic marks may contribute

significantly to the extent and timing of transcription from the viral genome and, consequently, to

the outcome of infection. Recent research has provided first insights into the viral and cellular

mechanisms that either maintain individual herpesvirus chromatin states or mediate transition

between them. Here, we summarise and discuss both early work and new developments pointing

towards common principles pertinent to the dynamic structure and epigenetic regulation of

herpesvirus chromatin. Special emphasis is given to the emerging similarities in nucleosome

assembly and disassembly processes on herpes simplex virus type 1 and human cytomegalovirus

genomes over the course of the viral productive replication cycle and during the switch between

latent and lytic infectious stages.

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INTRODUCTION

Chromatin structure and dynamics

Eukaryotic genomes are organised as chromatin, a highly dynamic complex principally composed

of DNA and histone proteins [1]. The fundamental repeating unit in chromatin is the nucleosome,

which is composed of 147 base pairs of DNA wrapped 1.65 turns around an octameric protein core

containing two copies each of the four histones H2A, H2B, H3, and H4 [2] (Figure 1). The linker

histone H1 binds to the DNA region between the nucleosomes to stabilise the chromatin fiber. The

packaging of DNA into chromatin ensures genome compaction and stability, but at the same time

creates a barrier for the access of protein (and RNA) factors to the underlying nucleotide

sequences. Thus, chromatin structure imposes profound effects on basically all DNA-templated

processes in eukaryotic cell biology including transcription. Major mechanisms modulating

chromatin structure and function include DNA methylation at CpG dinucleotides, covalent histone

modifications, histone variant exchange, and nucleosome remodelling including histone

displacement (eviction) (reviewed in [3]).

An extensive array of posttranslational, covalent modifications on histones has been

discovered, most of which occur at the amino-terminal protein tails (reviewed in [4]). Depending

on the nature and position of the modification, they are linked to different gene activities. For

example, acetylation of histone H3 on lysines 9 and 14 is coupled to active transcription (Figure

1). Histone acetylation and deacetylation are highly dynamic processes that are controlled by the

counteracting activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs).

Histone methylation catalysed by histone methyltransferases (HMTs) is believed to be a more

stable epigenetic mark and, depending on the position of the modification, can be associated with

either transcriptional activation or repression (Figure 1). Certain histone modifications including

acetylation cause changes in the net charge of nucleosomes, which can loosen DNA-histone

interactions [5].

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Covalent histone marks can also serve as binding sites for other proteins including

components of chromatin remodelling complexes (e.g., [6-8]), which are multi-subunit machines

that utilise ATP to alter histone-DNA contacts. The consequences of remodelling are unwrapping

of DNA, repositioning of nucleosomes, and/or histone eviction (reviewed in [3]). Among the best

characterised chromatin remodellers is the SWI/SNF complex initially identified in mating type

switching (SWI) and sucrose non-fermenting (SNF) yeast strains. Mammalian SWI/SNF

complexes contain the Brahma (BRM), Brahma-related gene 1 (BRG1) and eight to ten additional

subunits including the SNF5/integrase interactor 1 (Ini1) protein (reviewed in [9]).

Other key regulators of chromatin dynamics are histone chaperones. These proteins shield

the positive charge of histones and coordinate their exchange onto DNA during nucleosome

assembly and disassembly processes (reviewed in [10]). A DNA replication-coupled pathway

accounts for the bulk of normal cellular nucleosome assembly (reviewed in [11]). The chromatin

assembly factor 1 (CAF1) multisubunit chaperone, which is tethered to the replication processivity

clamp (proliferating cell nuclear antigen), cooperates with other histone chaperones such as anti-

silencing function 1 (ASF1) to mediate histone positioning at the replication fork. In addition to

the replication-coupled process, replication-independent nucleosome assembly pathways involving

a distinct but overlapping set of protein factors including variant histones have also been described

(reviewed in [11]).

Herpesvirus classification and life cycle

The Herpesviridae are a large family of ubiquitous viruses infecting diverse vertebrate species

including humans. There are currently eight known human herpesviruses classified in three

subfamilies (-, -, and -Herpesvirinae). The human -herpesviruses encompass HSV-1 or

human herpesvirus 1 and HSV-2 or human herpesvirus 2 as well as VZV or human herpesvirus 3.

The -herpesviruses are represented by human cytomegalovirus (hCMV or human herpesvirus 5),

human herpesvirus 6 and human herpesvirus 7. Finally, EBV or human herpesvirus 4 and Kaposi’s

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sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) form the human -herpesvirus

subfamily [12].

Herpesviruses vary greatly with respect to the cell types infected and the diseases they

cause, yet they share common structural features (reviewed in [13]). Each herpesvirus virion

contains a single, linear, double-stranded DNA molecule of 120,000 to 240,000 nucleotides

packaged in an icosahedral capsid. The capsid is surrounded by a multi-protein coat, known as the

tegument, and a lipid bilayer in which viral glycoproteins are embedded. Common to all

herpesviruses is the establishment of life-long latent infections after a phase of lytic replication.

During the lytic infectious cycle most herpesvirus genes are expressed resulting in progeny virion

production and viral spread. In contrast, gene expression from the viral DNA is largely repressed

during latency, and virus particles are not formed. Herpesvirus reactivation from latency in

response to any of a number of external triggers results in recurrent infections.

The herpesvirus replication cycle begins with binding of the virion to the respective cell

surface receptor and entry into the host cell cytoplasm. Viral capsids are then rapidly transported

to the nuclear pore, and the linear viral DNA is released into the cell nucleus where it may

circularise. Some components of the viral tegument are transported independently to the nucleus,

including HSV-1 virion protein (VP) 16 (also known as -transinducing factor) and hCMV

phosphoprotein pp71 (encoded by the unique long [UL] 82 open reading frame), which are potent

transcriptional activators of the viral immediate-early (IE) genes. The IE proteins, in turn,

counteract intrinsic and innate host defence mechanisms and activate the early class of genes

whose products are typically involved in the process of viral DNA replication. With the onset of

DNA replication, the late viral proteins are expressed, most of which are structural constituents of

the virus particle. Viral transcription, DNA replication, and nucleocapsid assembly proceed within

specialised intranuclear replication compartments that develop from structures known as

promyelocytic leukemia (PML) bodies or nuclear domain 10 (ND10). Finally, the nucleocapsids

acquire tegument proteins, envelope during sequential budding through intracellular membranes,

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and are released to the extracellular space from where they can infect surrounding cells (reviewed

in [13]).

Herpes virology meets chromatin biology

From the beginnings of molecular herpes virology, it was recognised that the intimate relationship

between these nuclear replicating DNA viruses and their host cells almost certainly has to include

cellular chromatin pathways. The early interest in herpesvirus DNA chromatinisation during the

1970s and 1980s was followed by a period of astonishing neglect, which lasted up until recently.

However, concurrent with technological advances in chromatin immunoprecipitation (ChIP)

techniques, there has been renewed and increasing interest in herpesvirus chromatin biology

within the last number of years. This review will summarise and discuss both early work and new

discoveries regarding the structural dynamics and epigenetic control mechanisms of herpesvirus

chromatin. The enormous recent progress in deciphering how covalent core histone modifications

regulate herpesvirus chromatin function during lytic and latent infection has already been

accounted for in several excellent current review articles [14-19]. We will therefore focus this

present review on findings related to the dynamics of nucleosome occupancy on HSV-1, hCMV

and other herpesvirus genomes rather than changes in histone modifications, although histone

acetylation will be discussed if relevant in this context.

THREE GLOBAL CHROMATIN STATES OF HERPESVIRUS DNA

Chromatin-free state of encapsidated viral DNA

Viruses usually package their genomes at high density in order to make efficient use of the limited

space available within the viral capsid. In many cases nucleic acid compaction and neutralisation

of negative charge is accomplished by complexing the viral genome with basic proteins. For

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example, the linear double-stranded DNA genomes of adenoviruses are packaged with viral core

proteins, including the histone H3-related protein VII, in a chromatin-like structure [20-23]. The

genomes of other small DNA viruses, namely papilloma- [24] and polyomaviruses [25, 26], are

typically associated with host-derived nucleosomes when packaged in viral capsids. However,

since proteins occupy a significant fraction of the available space, their presence reduces the

overall packing efficiency.

From early on, the large DNA genomes of herpesviruses were believed to be free of

histones when encapsidated in the virion and no other abundant viral or cellular protein intimately

coating the viral DNA has been identified [27-29]. Instead, HSV-1 virions contain polyamines at

fixed molar ratios relative to the viral genome, and it was suggested that spermine serves to charge

neutralise at least 40% of the DNA [30]. Interestingly, polyamines have also been shown to be

present in the heads of icosahedral bacteriophages, where the DNA duplexes are close packed in a

liquid crystalline state with little or no protein (reviewed in [31]). Indeed, encapsidated HSV-1

DNA appears to exhibit a liquid crystalline structure closely resembling that in bacteriophages T4

and λ [20]. The view of histone-free herpes virion DNA has gained support by more recent work,

including western blotting for histone H3 on purified hCMV and HSV-1 capsids ([32, 33]; A.

Nitzsche and C. Paulus, unpublished results). Furthermore, global mass spectrometry-based

proteome analyses of hCMV [34], EBV [35], and KSHV [36, 37] particles did not identify

histones or other proteins typically associated with chromatin, although a variety of cellular non-

chromatin proteins were present (reviewed in [38]). Curiously, mass spectrometry carried out on

murine cytomegalovirus (mCMV) particles identified H2A, despite the fact that none of the other

core histones was detected [39].

In sum, the available evidence largely excludes the possibility that nucleosomes are

associated with encapsidated herpesvirus genomes. Rather, the large size of herpesvirus genomes

demands highly efficient DNA packaging with few if any proteins.

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Regular nucleosomal viral chromatin structure during latent infection

During latency, herpesvirus genomes typically persist as circular episomes in the nuclei of certain

host cells. The latent viral episomes appear to be predominantly associated with histones that form

regular nucleosomal arrays resembling cellular chromatin. This has been experimentally

demonstrated for EBV (e.g., [40-42]), HSV-1 (e.g., [43]), and KSHV (e.g., [44]). It likely also

holds true for other members of the herpesvirus family, including hCMV, where formal proof for a

regularly nucleosomal state of latent viral DNA is still missing. During latency, the nucleosomes

on lytic viral genes generally display histone modification patterns indicative of heterochromatin,

as has been reviewed elsewhere [14-19, 45]. Thus, latent herpesvirus chromatin appears to exist in

an epigenetically repressed state that precludes transcription from large parts of the viral

chromosome.

On the other hand, a limited number of active regions in the latent herpesvirus episomes

may be more irregularly chromatinised or even histone-free. For example, Wensing et al. found

experimental evidence for low nucleosome density in the region encompassing the origin of latent

DNA replication (oriP) and the latency-associated EBV-encoded small RNA (EBER) genes in

EBV-carrying cells [41]. Alternatively or additionally, nucleosomes on viral latency genes may

carry covalent histone marks associated with active transcription. H3 hyperacetylation has, for

example, been shown for the promoter and enhancer of the HSV-1 latency-associated transcript

(LAT) [46] and for the KSHV latent origin of DNA replication [44] during viral latent infection.

The question of how eu- and heterochromatin domains are kept separate on the latent viral

chromosome is currently being addressed by examining “insulator” elements (e.g., CCCTC sites)

on the viral DNA, which bind proteins (e.g., CCCTC-binding factor) that maintain chromatin

boundaries (reviewed in [14]).

Irregular chromatinisation of viral DNA during lytic infection

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The general state of lytic herpesvirus chromatin has been a matter of debate reaching back to the

1970s. A series of studies provided evidence for an exclusive or at least highly predominant non-

nucleosomal structure of intranuclear HSV-1 [29, 47-51] and EBV [40] genomes in various

productively infected cell types. Furthermore, it has been reported that HSV-1 DNA accumulates

in replication compartments that exclude histones H1 and H2B [52, 53]. Nonetheless, others did

find evidence for significant association of HSV-1, HSV-2, or hCMV DNA with nucleosomes and

for the presence of core histones at the intranuclear sites of hCMV genome accumulation during

productive replication [54-59]. Moreover, numerous recent ChIP studies have revealed that all

four core histone classes, including posttranslationally modified forms, associate with HSV-1 and

hCMV genomes inside productively infected cells (e.g., [32, 33, 58-72]). However, ChIP assays

also demonstrated that core histones are generally underrepresented on lytic HSV-1 and hCMV

genomes as compared to typical cellular genes [59, 66, 69].

We suppose that the seemingly conflicting reports about chromatin content of herpesvirus

DNA during viral productive replication may simply reflect variations in the starting conditions

and/or timing of infection. The proportion of histone-associated herpesvirus DNA may, for

example, vary with the input multiplicity used for infection ([65]; A. Nitzsche and C. Paulus,

unpublished results). Additionally, the herpesvirus chromatin structure may undergo temporal

changes over the course of the viral replication cycle. In support of this view, our recent work has

demonstrated a substantial (up to 10-fold) increase in global histone H3 and nucleosome

occupancy on viral genomes between the early and late stages of the hCMV replicative cycle [59].

This increase proved to be largely dependent on the process of viral DNA synthesis implying

replication-dependent nucleosome assembly mechanisms [59]. Most likely, a larger proportion of

chromatinised hCMV genomes exist after viral DNA replication (and prior to encapsidation) as

compared to the pre-replicative phase, where the number of naked viral DNA molecules may

exceed that of nucleosome-occupied genomes (Figure 2).

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The consensus deduced from these various observations is that the herpesvirus genomes

that have been studied so far are not assembled in a regular array of nucleosomes during

productive infection. However, nuclear viral DNA molecules do partially associate with

nucleosomes. An intermediate state of herpesvirus genome chromatinisation may, on the one hand,

allow for almost unrestricted transcription factor access and preclude epigenetic silencing of viral

gene expression. On the other hand, the lytic viral chromatin state may still permit a certain extent

of nucleosome-based regulation. In fact, herpesvirus lytic genes typically acquire active

euchromatin-specific histone modifications over the course of the productive infectious cycle

generally correlating with timing of transcription from the viral genome (reviewed in [14-19]).

HERPESVIRUS CHROMATIN ASSEMBLY

Consecutive DNA replication-independent and replication-coupled viral chromatin

assembly during lytic infection

As discussed above, the uncoated herpesvirus genome released into the host cell nucleoplasm is

believed to be initially “naked” with respect to chromatin proteins. However, it is clear now that

host-derived core histones rapidly associate with herpesvirus genomes during or very soon after

nuclear entry. In fact, histone H3 has been found associated with intranuclear hCMV DNA as

early as 30 minutes post virus inoculation [59]. Presumably, the cellular nucleosome assembly

machinery targets herpesvirus genomes entering the nucleus. In agreement with this assumption,

the histone chaperones CAF1 p48 and ASF1 were found co-localised with or juxtaposed to the

nuclear sites of hCMV parental genome deposition [59].

A general feature of nuclear replicating DNA viruses, including members of all herpesvirus

subfamilies, is the preferential association of their parental genomes and pre-replicative sites with

ND10 (reviewed in [73]). Viral DNA may associate with pre-existing ND10, but it has also been

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shown that structures resembling ND10 form de novo in association with viral genome complexes

during the initial stages of HSV-1 infection [74]. Interestingly, the ND10 constituents PML,

Sp100, death domain-associated protein (Daxx), and the SWI/SNF family member -

thalassemia/mental retardation syndrome X-linked protein (ATRX) are all involved in an intrinsic

anti-viral response against HSV-1 and/or hCMV that limits viral gene expression [75-80].

Moreover, several ND10 proteins have been implicated in epigenetic processes (reviewed in [81]).

For example, Daxx interacts with histones as well as HDACs and forms part of a chromatin

remodelling complex together with ATRX [82, 83]. Therefore, it appears likely that ND10

proteins contribute to formation of a repressive chromatin structure on newly infecting virus DNA.

Based on inhibitor studies [59] and the notion that viral DNA replication does not start

until several hours (~ 6 h in HSV-1 and ~24 h in hCMV) post infection, primary viral genome

chromatinisation very likely occurs exclusively via DNA replication-independent nucleosome

assembly processes. However, following the pre-replicative phase of primary nucleosome

deposition, hCMV genomes eventually undergo DNA replication-dependent nucleosome assembly

[59]. Evidence for consecutive replication-independent and replication-coupled stages of viral

genome chromatinisation has also been provided for HSV-1. For instance, early HSV-1 chromatin

predominantly contains the histone variant H3.3 [84], which is incorporated by replication-

independent mechanisms [85]. In contrast, canonical H3.1 was only found in late HSV-1

chromatin and its incorporation was dependent on viral DNA synthesis [84]. Replication-coupled

nucleosome deposition may result in substantially increased amounts of chromatinised nuclear

herpesvirus DNA (see text above and Figure 2) with potential implications for viral late gene

expression and the process of progeny genome encapsidation (see text below and Figure 2).

Transition to latent viral chromatin

It is an exciting but unresolved question whether transition to the latent herpesvirus chromatin

state involves only replication-independent nucleosome assembly mechanisms or if it has to

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proceed through at least one round of DNA replication. Irrespective thereof, most herpes

virologists would probably argue that folding of viral chromatin into a regular array of

nucleosomes during establishment of latency is predominantly or exclusively determined by the

host cell. Nevertheless, the KSHV latency-associated nuclear antigen (LANA), besides being

essential for viral episome maintenance, may have an active role in facilitating latent chromatin

formation. In particular, LANA is unprecedented in its ability to promote chromatin condensation

via interaction with histones H2A-H2B [86, 87]. In HSV-1, LAT functions differently but to

apparently similar ends by promoting heterochromatin assembly on viral lytic genes [88, 89]. A

latency promoting activity has also been recently identified in hCMV [90], but the corresponding

protein does not seem to be involved in chromatin regulation [91]. Thus, cellular and viral factors

apparently cooperate in the chromatin-related events that accompany establishment of viral latency

in some if not all herpesviruses.

HERPESVIRUS CHROMATIN DISASSEMBLY

Maintenance of intermediate viral genome chromatinisation during lytic infection

We are at the very outset of understanding the mechanisms accounting for the relatively low levels

of nucleosomes on herpesvirus genomes during productive replication as compared to latency.

Conceivably, viral chromosomes may have differential access to the cellular nucleosome

deposition machinery during the lytic versus latent phase of infection. Indeed, there is evidence

that the spatial intranuclear distribution of herpesvirus genomes during productive replication

differs substantially from that in quiescently infected cells (e.g., [92, 93]).

Another attractive explanation for the low degree of herpesvirus chromatin content during

productive infection is provided by the idea that viral gene products may counteract deposition or

promote eviction of histones on nuclear viral DNA. These gene products (proteins and/or RNAs)

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may be present and active during productive infection (and reactivation from latency) and absent

or inactive during the latent phase. In fact, several very recent reports support the scenario that

certain herpesvirus proteins are critical to keep down the nucleosome load on lytic viral genomes.

In the following paragraphs we will introduce these proteins and discuss how they may negatively

affect nucleosome occupancy on lytic (and latent) herpesvirus genomes.

HSV-1 VP16

The tegument protein VP16 is the main transcriptional activator of HSV-1 IE genes and has served

as a prototype transcriptional activator for decades [94]. By studying a mutant virus lacking the

VP16 activation domain, Herrera and Triezenberg provided the first direct evidence that a

herpesvirus gene product can have an active role in reducing the amount of core histones

associated with viral DNA [66]. Histone H2A, H2B, H3, and H4 occupancy increased

considerably at all tested viral loci and sometimes even reached levels comparable to a cellular

gene in VP16 mutant as opposed to wild-type infections [66, 69]. Interestingly, the VP16

activation domain has also been shown to induce large-scale chromatin decondensation when

targeted to cellular DNA [95-97]. VP16 not only recruits a multitude of basal transcription factors

and transcriptional co-activators (including the following HATs: p300, cAMP response element

binding-binding protein [CBP], p300/CBP-associated factor [PCAF], and general control non-

derepressible 5 [GCN5]; reviewed in [16]), but also at least two components of the human

SWI/SNF chromatin remodelling complex, namely BRG1 and BRM [66]. Binding of SWI/SNF to

the VP16 activation domain leads to eviction of nucleosomes from chromatin templates in vitro

[98]. Collectively, these observations point to a straight-forward model in which histone

deposition on HSV-1 genomes is partially reversed through the activities of chromatin modifying

(including nucleosome remodelling) proteins that become recruited to viral chromosomes by

VP16. This model would nicely link the chromatin-targeted effects of VP16 to transcription from

the viral genome. Suprisingly, however, RNA interference of BRM, BRG1, p300, CBP, PCAF,

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and/or GCN5 (either individually or in certain combinations) during productive HSV-1 infection

did not affect viral IE gene expression [99]. Therefore, the role of VP16 in transcriptional

activation of HSV-1 genomes appears to be more complex than commonly thought.

HSV-1 VP22

VP22 is another abundant HSV-1 tegument protein that has been implicated in viral genome

chromatinisation. VP22 binds to template activating factor 1 (TAF1) histone chaperones. The

TAF1 proteinsoriginally identified as host factors promoting adenovirus DNA replication, bind to

core histones and have a role in chromatin decondensation and remodelling (reviewed in [100]).

Interestingly, VP22 inhibits nucleosome deposition on naked DNA and might thus interfere with

primary HSV-1 genome chromatinisation [101].

HSV-1 ICP0

Kutluay and Triezenberg demonstrated very recently that not only virion components but also one

or more IE proteins of HSV-1 contribute to reducing the histone load on viral genomes during

lytic replication [69]. Shortly beforehand, Cliffe and Knipe reported that the HSV-1 IE gene

product infected cell protein (ICP) 0 promotes histone H3 removal from viral IE (ICP4) and E

(ICP8) gene promoters during productive infection [65]. A role for ICP0 in reducing global histone

association of HSV-1 DNA is consistent with the fact that the protein stimulates transcription from

all kinetic classes of viral promoters [102, 103]. ICP0 binds to several HDACs [104] as well as to

the repressor element 1-silencing transcription factor (REST)/co-repressor to REST

(CoREST)/HDAC complex [105, 106]. Likewise, the bovine herpesvirus type 1 (BHV-1) ICP0

orthologue (bICP0) interacts with HDAC1 and p300 [107, 108]. Moreover, HSV-1 ICP0 has been

shown to inhibit histone deacetylation [109] and/or to promote histone acetylation [65, 110]. In

some experimental settings, the replication defects of ICP0 mutant viruses can also be

complemented, at least in part, by chemical HDAC inhibitors [109, 111, 112]. In addition to

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serving as a binding site for chromatin remodelling proteins (e.g., [6-8]), histone acetylation

changes the net charge on nucleosomes, which may loosen histone-DNA interactions [5]. This

idea is supported by the observation that acetylated histones are easier to displace from DNA both

in vivo [113] and in vitro [6, 7, 114]. Thus, ICP0 may act indirectly to decrease chromatin content

on viral DNA by increasing histone acetylation levels. However, the idea that ICP0 could activate

viral transcription via histone acetylation remains controversial. In fact, Everett et al. have

demonstrated that HDAC inhibition fails to enhance plaque formation by an ICP0-null mutant

HSV-1 in human fibroblasts and hepatocytes [115], and the viral protein does not seem to have

global effects on histone acetylation [104]. Alternatively, since ICP0 is a E3 ubiquitin ligase [116,

117], one could hypothesise that the viral protein might cause degradation of histones or non-

histone proteins involved in chromatin assembly. In fact, ICP0 has been shown to promote

proteolysis of centromere protein A (CENP-A), a histone H3 variant, and other kinetochore

proteins [118-120]. Additionally, ICP0 induces degradation of PML, thereby disrupting ND10 and

dispersing their constituent proteins including Sp100 and Daxx [121-125]. Although this

observation may provide further evidence for a role of ND10 in herpesvirus chromatin assembly,

the mechanism by which ICP0 promotes histone removal from viral chromosomes remains to be

determined.

Intriguingly, the reactivation of viral genomes from at least a subset of latently infected

neurons in vivo and quiescently infected cells in vitro can be promoted by ICP0 [126-128]. Given

that a regular array of nucleosomes is present on the HSV-1 genome in latently infected neurons, it

is possible that a function of ICP0 in reactivation from latency involves the reduction in histone

association with the viral genome. In fact, the absence of ICP0 correlates with increased

nucleosome abundance on quiescent HSV-1 genomes [129]. On the other hand, a recent study by

Coleman et al. found no decrease in histone association with the viral genome following ICP0-

mediated derepression in vitro [110]. However, this does not rule out the possibility that ICP0

mediates removal of histones from latently infected neuronal cells.

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HSV-1 ICP8

The HSV-1 early protein ICP8 has been proposed to recruit histone modifying and chromatin

remodelling complexes, including histone chaperones and components of SWI/SNF (e.g., BRG1

and BRM), into viral replication compartments and onto progeny viral DNA [130]. ICP8 can

activate late HSV-1 gene expression independent of its role in stimulating viral DNA replication

[131], and this might be due to a reduction of chromatin on viral DNA [16].

VZV IE63

The VZV IE63 protein has been shown very recently to interact with histone chaperone ASF1

[132] in both lytic and latently infected neurons. IE63 increased the binding of ASF1 to histones

H3.1 and H3.3, which may have consequences for histone deposition on nuclear VZV genomes.

Other herpesvirus proteins

To our knowledge, no - or -herpesvirus protein globally affecting the nucleosome content of

viral chromatin has so far been identified. However, there are several suspects. In fact,

components of the nucleosome remodelling and deacetylase (NuRD) protein complex (e.g., CAF1

p48, HDAC1 and HDAC2) were shown to co-purify with the hCMV early protein pUL29/28

[133]. Besides, the hCMV IE1 protein of Mr 72,000 shares a number of functional features with

HSV-1 ICP0 including the ability to activate transcription, to disrupt ND10 in a PML-dependent

fashion (reviewed in [134]), and to antagonise histone deacetylation via HDAC interaction [63].

The mCMV IE1 ortholog also interacts with at least one HDAC [135] and with Daxx [136].

Furthermore, the hCMV and mCMV IE1 proteins have long been known to associate with

(mitotic) chromatin [137] or core histones [138, 139], respectively. Therefore, it is very tempting

to speculate that IE1 may have a role resembling that of ICP0 in globally counteracting viral

genome chromatinisation.

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Another candidate CMV chromatin regulator is the pp71 protein, which serves as a major

tegument transactivator with functional similarities to HSV-1 VP16 [140, 141]. Among other

activities, pp71 interacts with Daxx and antagonises Daxx-mediated histone deacetylation at the

viral major IE promoter by targeting the cellular repressor protein for degradation [78, 142-144].

Furthermore, pp71 displaces ATRX from ND10 at very early times post hCMV infection [80].

However, possible effects of pp71 on histone occupancy of hCMV genomes have not yet been

studied.

It is quite imaginable that -herpesvirus gene products that functionally resemble the HSV-

1 chromatin remodelling proteins, like the EBV BamHI Z left frame 1 (BZLF1) or the ORF

K8/KSHV basic leucine zipper (K-Zip) protein (both similar to ICP0 and IE1) may have a role in

limiting global viral DNA-nucleosome association during productive infection by their respective

viruses. Beyond that, two EBV nuclear antigens (EBNA) have been implicated in regulating

nucleosome occupancy on viral genomes locally. EBNA1 interacts with and destabilises

nucleosomes at the oriP, and it was suggested that this effect is important for origin activation

[145]. EBNA1 also exhibits general chromatin binding activity [146, 147], which is required for

intranuclear maintenance and non-stochastic segregation of latent viral episomes, and this protein

can disrupt ND10 [148]. EBNA2 mediates transcriptional activation of viral and cellular genes.

Among numerous host cell proteins, EBNA2 also binds to SNF5/Ini1, a component of the human

SWI/SNF complex [149]. It was postulated that the viral protein engages SWI/SNF to generate an

open chromatin conformation at EBNA2 responsive target genes [149].

A number of additional herpesvirus proteins have been shown to interact with host cell

chromatin components including histones, histone chaperones, nucleosome remodelling proteins,

and histone modifying enzymes. Several of these viral proteins have known effects on

posttranslational histone tail acetylation, methylation or phosphorylation. A comprehensive list of

these proteins and their associated histone-directed activities is presented as Table 1.

19

Viral chromatin disassembly during DNA packing into progeny capsids?

Given that herpesvirus genomes exist in a nucleosome-associated state inside the host cell nucleus,

how can it be that all progeny viral DNA packaged within virions is entirely devoid of histones?

There are two basic (not mutually exclusive) explanations to this problem [59]: (1) viral DNA

destined for incorporation into capsids does not undergo nucleosome association or (2)

chromatinised progeny DNA is subject to complete nucleosome eviction before or during

encapsidation.

In support of possible explanation (1), a study by Oh and Fraser claimed that newly

synthesised HSV-1 DNA lacks histone H3 [32]. However, others have observed that the total

amount of HSV-1 and hCMV DNA associated with core histones increases with viral DNA

replication [59, 65, 69], and it was suggested that both naked and chromatinised HSV-1 genomes

may co-exist in the late stages of viral replication [65]. In this scenario, the histone-free fraction of

viral genomes may represent either packaged DNA or DNA destined for packaging. It is difficult

to conceive how a significant proportion of progeny viral genomes would escape the cellular

replication-dependent nucleosome assembly machinery to stay completely histone-free [59].

Potentially, chromatin assembly proteins may become overburdened and/or free histone pools may

be depleted towards the end of infection. At least in -herpesviruses, the latter could be a possible

consequence of the host shut-off function.

Following explanation (2), active disassembly of hCMV chromatin may occur before or

during packaging of replicated viral DNA into progeny capsids ([59]; Figure 2). In fact, two recent

studies on the temporal dynamics of hCMV chromatin assembly and modification found a specific

decrease in histone H3 occupancy at all tested viral genomic regions during very late times (72 to

96 hours) post infection [33, 59] consistent with the idea of nucleosome eviction as a prerequisite

for herpesvirus DNA packing. Interestingly, there is some precedent for partial viral chromatin

disassembly in simian virus 40 (SV40) and other polyoma viruses, which carry core histones

during virion encapsidation but lose their association with other chromatin proteins, including

20

linker histones and high mobility group proteins [150]. Provided that the chromatin disassembly

hypothesis is correct, we anticipate that the activities of unidentified viral and/or cellular proteins

(potentially including proteins listed in Table 1) forming ATP-utilising chromatin remodelling

complexes on progeny viral DNA would almost certainly be required to successfully complete the

herpesvirus infectious cycle.

CONCLUSIONS AND PERSPECTIVES

While it has long been accepted that herpesvirus genomes exist as naked DNA inside virus

particles or folded into regularly spaced nucleosomes in latently infected cell nuclei, the state of

nuclear herpesvirus DNA in productively infected cells remained controversial. Previous evidence

of non-nucleosomal lytic herpesvirus DNA is increasingly superseded by the view that a

substantial proportion of viral genomes become assembled with nucleosomes during productive

infection, albeit at much lower density compared to typical cellular genes or latent viral chromatin.

This intermediate state of herpesvirus genome chromatinisation may permit relatively unrestricted

transcription factor access and preclude transcriptional silencing via heterochromatin formation (as

typical for latent infections) while simultaneously allowing for a certain extent of nucleosome-

based regulation.

We anticipate that, in the near future, our rudimentary knowledge about the nucleosome-

directed mechanisms that mediate between the highly dynamic herpesvirus chromatin states will

quickly expand. It emerges that nucleosome occupancy coupled with core histone modifications

and other epigenetic mechanisms (such as histone variant exchange) may be fundamental to

transcriptional regulation of the viral genome during both lytic and latent infection as well as in

the switch between these two infectious stages. However, future research will likely move beyond

the transcription-centred view of viral chromatin, and we may learn about nucleosome-based

effects on other fundamental DNA-associated processes in herpesvirus infection such as

21

subnuclear deposition, repair, recombination (including circularisation), replication and

encapsidation of viral genomes. Finally, epigenetic pathways will hopefully provide new

opportunities for the development of anti-viral compounds that may outperform existing drugs in

the prevention or treatment of human herpesvirus infection and disease.

22

ACKNOWLEDGEMENTS

We apologise to our many colleagues whose work could not be cited owing to space limitations.

We thank Roger Everett and Hans Helmut Niller for very helpful comments on the manuscript,

Felicia Goodrum and Eran Segal for insightful discussions, and Hans Wolf for continuous support.

MN is supported by grants from the Deutsche Forschungsgemeinschaft (NE 791/2-2), the

European Union (TargetHerpes; LSHG-CT-2006-037517), and the Human Frontier Science

Program (RGY71/2008).

23

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FIGURE LEGENDS

Figure 1. Chromatin structure and regulation. At the top, the normal primary structure of

eukaryotic chromatin existing as an array of regularly spaced nucleosomes is schematically

presented. At this level, chromatin can be modified by nucleosome assembly or disassembly (de novo

assembly, exchange or eviction of nucleosomes). A nucleosome, shown at higher resolution in the

centre of the diagram, is composed of DNA wrapped around two copies each of the core histones

H2A, H2B, H3, and H4. Core histones are subject to posttranslational covalent modifications that

primarily affect their tail domains. Typical histone H3 tail modifications include lysine 9 and 14

acetylation (K9/14ac), lysine 4 di- or trimethylation (K4me2/3), and lysine 9 di- or trimethylation

(K9me2/3). Processes and modifications that generally correlate with active transcription are labelled

green, whereas processes and histone marks normally associated with transcriptional repression are

labelled red.

Figure 2. Global patterns of nucleosome occupancy on viral genomes during lytic (high multiplicity)

and latent herpesvirus infection. In its encapsidated state, the viral DNA is linear and nucleosome-

free (top of the diagram). After release into the host nucleus, herpesvirus genomes typically

circularise, and at least a subset of them undergo nucleosome deposition by DNA replication-

independent mechanisms. This process of primary herpesvirus chromatin assembly may be part of

an intrinsic anti-viral defence mechanism facilitated by cellular ND10 constituents and counteracted

by viral tegument proteins such as HSV-1 VP16 and/or VP22. Lytic viral genomes most likely

maintain an intermediate state of nucleosome occupancy throughout infection, which is optimised for

active transcription with some degree of chromatin-based control (e.g., via histone modifications).

However, at least in hCMV, the proportion of chromatinised viral genomes increases concurrent with

viral DNA synthesis (DNA replication-dependent nucleosome assembly). Consequently, nucleosome

disassembly may be required to permit packing of replicated viral DNA into progeny capsids.

Alternatively, DNA replication may result in both chromatinised and histone-free viral genome

populations only the latter of which becomes packaged without a requirement for nucleosome

disassembly (not shown). Finally, during transition into latency herpesvirus DNA acquires a full

40

41

complement of nucleosomes resulting in regularly chromatinised viral genomes prone to

heterochromatin formation and transcriptional silencing. This process may be counteracted by

herpesvirus proteins including HSV-1 ICP0, VP16, and/or VP22. Conversely, in the process of

reactivation from latency, nucleosome disassembly (potentially facilitated by viral proteins like HSV-

1 ICP0) must likely occur to permit transcriptional activation of viral lytic cycle genes. +, positive

effect; –, negative effect; ?, experimentally non-verified.

Table 1. Herpesvirus gene products affecting nucleosome-based chromatin modification

Interactions Effects

Herpesvirus Viral

protein/RNA Histones Histone

chaperones Remodelling

factors HATs HDACs HMTs

DNA/ chromatin

ND10 Histone

modifications Nucleosome occupancy

bICP0 – – – p300 HDAC1 – – – – – BHV-1

bVP22 H1

Core histones – – – – – – – – –

ICP0 (CENP-A) – – CBP (HDAC1, 2) HDAC4, 5, 7

– – + Acetylation↑ ↓

ICP8 – (CAF1 p48) SWI/SNF – HDAC2 – + – – –

LAT – – – – – – – – Methylation↑↓ –

US3 – – – – (HDAC1) – – – – –

VP16 – – BRG1 BRM

GCN5, p300 PCAF

– Set1 + – Acetylation↑ ↓

HSV-1

VP22 – TAF1 – – – – – – – ↓

-H

erpe

svir

uses

VZV IE63 – ASF1 – – – – – – – –

IE1 – – – – HDAC3 – + + Acetylation↑ –

IE2 – – – CBP, p300

PCAF HDAC1-3 HDAC4, 51

G9a SUV39H1

+ + Acetylation↑Methylation↑

–

pp71 – – Daxx – – – – + –2 – hCMV

pUL29/28 – (CAF1 p48) (Mi-2 – (HDAC1, 2) – – – – – -H

erpe

svir

uses

mCMV mIE1 Core histones – Daxx – HDAC2 – + + Acetylation↑ –

BGLF4 – – – – – – + – Phosphoryl.↑3 –

BZLF1 – – – CBP – – + + Acetylation↑ –

EBNA1 + – – – – – + + – ↓ (local)

EBNA2 – – BRG1

SNF5/Ini1 CBP, p300

PCAF HDAC4 – + – Acetylation↑ –

EBNA3C – – – p300 HDAC1, 2 – – – – – -H

erpe

svir

uses

EBV

EBNA5 – – – – HDAC4 – – + – –

LANA H2A-H2B – – CBP – SUV39H1 + + – –

ORF50/ K-RTA

– – BRG1 CBP HDAC1 – – – – –

ORF K8/ K-bZip

– – SNF5/Ini1 CBP – – + + – – KSHV

ORF K9/ vIRF-1

– – – CBP, p300 – – – + – – -H

erpe

svir

uses

MHV68 ORF36 – – – – – – – – Phosphoryl.↑3 –

BGLF4, EBV BamHI G left frame 4 protein; K-RTA, KSHV replication and transcription activator; MHV68, murine -herpesvirus 68; Set1, SET domain containing family member 1; SUV39H1, suppressor of variegation 3-9 homologue 1; US, unique short; vIRF-1, viral interferon regulatory factor 1; +, positive; –, negative or unknown; ↑, up-regulation; ↓, down-regulation. Protein designations in brackets indicate indirect or unconfirmed interactions. For references, see text and [14-19]. 1 C. Paulus, unpublished results. 2 hCMV pp71 may promote histone acetylation by targeting Daxx-HDAC complexes. 3 EBV BGLF4 and MHV68 ORF36 kinases stimulate phosphorylation of the variant histone H2A.X.

HistoneModification

Nucleosome(Dis)Assembly

+

K9me2/3K9/14ac

K4me2/3

H3H4

H2AH2B

Paulus et al., Figure 1

Paulus et al., Figure 1

Lytic CycleViral transcription globally on

Lytic genes irregularly nucleosome-occupiedand euchromatic

? Disassembly Assembly(replication-independent)? ND10 +? VP16 -? VP22 -

LatencyViral transcription globally off

Lytic genes regularly nucleosome-occupiedand heterochromatic

Reactivation? ICP0 +

VP16 -

ICP0 -? VP22 -

Assembly(replication-dependent)

? ND10 +

Paulus et al., Figure 2

Paulus et al., Figure 2