Chromatin structure regulates human cytomegalovirus gene expression during latency, reactivation and lytic infection

Biochimica et Biophysica Acta 1799 (2010) 286–295

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g r m

Chromatin structure regulates human cytomegalovirus gene expression during latency, reactivation and lytic infection John Sinclair ⁎ University of Cambridge, Department of Medicine, Level 5, Addenbrooke's Hospital, Hills Rd, Cambridge, CB2 2QQ, UK

a r t i c l e

i n f o

Article history: Received 26 March 2009 Received in revised form 2 July 2009 Accepted 1 August 2009 Available online 12 August 2009 Keywords: Cytomegalovirus Latency Reactivation Chromatin Histone modification Lytic infection Transcriptional regulation

a b s t r a c t Infection of cells with human cytomegalovirus (HCMV) has two potential outcomes. For instance, infection of fibroblasts results in extensive viral gene expression, viral DNA replication and release of progeny virus. In contrast, in undifferentiated myeloid cells, the lytic transcription programme of HCMV is effectively suppressed and cells undergo latent infection. It is now accepted that the suppression of viral lytic gene expression observed during latency in myeloid cells is a result of the inability of undifferentiated cell types to support robust viral immediate early (IE) gene expression — crucial genes responsible for driving the lytic cycle. The repression of IE gene expression in undifferentiated myeloid cells, at least in part, results from specific post-translational modifications of histones associated with the viral major immediate early promoter (MIEP). In cells of the early myeloid lineage, the histone modifications present on the MIEP impart on it a repressive chromatin structure preventing transcriptional activity. Reactivation of HCMV lytic infection is correlated to changes in histone modifications around the MIEP resulting in a chromatin structure conducive to transcriptional activity. These changes are intimately linked with the differentiation of myeloid cells — a phenomenon known to reactivate latent virus in vivo. Chromatin structure of the viral MIEP, therefore, plays a crucial role in latency and reactivation of this persistent human herpesvirus. Whether chromatin-mediated regulation of viral lytic gene expression also occurs, is only beginning to be addressed. However, recent work suggests that all classes of lytic HCMV promoters are subjected to regulation by post-translational modification of their associated histones throughout the time course of infection. Incoming viral genomes appear to be the targets of intrinsic cellular defence mechanisms which attempt to silence viral gene expression through chromatinisation. Viral functions eventually overcome these cellular repression mechanisms permitting high levels of IE gene expression which results in modification of the chromatin structure of early and late gene promoters driving a regulated cascade of viral lytic gene expression and virus production. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction The herpesviridae family can be sub-divided into three subfamilies termed alpha, beta, and gamma; largely based on their biological properties and on their genome sequence and structure. Different subfamily members can have quite disparate biological properties but one common property of all herpesviruses is their ability to persist for the lifetime of the host. This plays a crucial role in the long-term carriage of these human pathogens in vivo. Human cytomegalovirus (HCMV) is the prototypic member of the Betaherpesvirinae subfamily that is characterised by strict species specificity, a very long replication cycle, the ability to cause cell enlargement (cytomegaly) and persistence in cells of the myeloid lineage [1].

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The 230 kb genome of HCMV encodes around 200 genes [2,3]. Like all herpesviruses, lytic infection results in a regulated cascade of viral gene expression resulting from extensive transcription of the viral genome which drives viral DNA replication and production of infectious virions. The transcription programme accompanying HCMV lytic infection starts with immediate early (IE) gene expression followed by expression of early (E) and finally late (L) genes: IE gene expression, exemplified by the major IE72 and IE86 gene products of HCMV, is crucial for the induction of E and L viral gene expression and IE72/IE86 also target numerous cell functions to optimise the cellular environment for high levels of viral DNA replication and virus production [4–8] (Fig. 1A). After a primary infection, HCMV persists for the life time of the host. This persistence likely involves sites in the host which continually undergo low level productive infection, but also involves sites which undergo latent infection. During latency, the lytic transcription programme is suppressed and viral transcription is restricted to expression of a few latency-associated transcripts [1].

1874-9399/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2009.08.001

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Fig. 1. Transcription of the HCMV major IE gene products IE72 and IE86 expressed during productive infection is driven by the complex major IE promoter/enhancer (MIEP). A) The most abundant HCMV proteins expressed at IE times of infection are the major IE gene products, IE72 and IE86 which result from differential splicing of the same primary transcript; IE72 comprising exons 2, 3 and 4 and IE86 comprising exons 2, 3 and 5. IE72 and IE86 act synergistically to activate viral early (E) and late (L) gene expression and IE86 can also negatively auto-regulate its own promoter by binding to the cis-repression signal (crs). These viral products also have profound effects on cellular functions such as cell cycle and cell transcription. B) Expression of these major IE proteins is driven by the viral major IE promoter (MIEP) which comprises a core promoter, enhancer, unique and modulator region. Within the enhancer, binding sites for known cellular transcription factors have been identified (see text for details). NF-κB, CREB/ATF and YY1/ERF bind to the 18 bp, 19 bp and 21 bp repeats, respectively. The transcription start site is designated by the forward arrow at + 1. Negative regulatory factors are highlighted by the blue background. Reproduced with permission from [26].

Consequently, the viral genome is maintained and carried in the absence of production of infectious virus. Importantly, this latent virus can routinely reactivate in vivo. The ability of HCMV to persist is undoubtedly crucial for its success as a human pathogen. HCMV causes widespread persistent infection in the human population with 90% of some populations seropositive for virus. Although a primary infection with HCMV rarely results in clinical disease, primary infection in the immunocompromised or immuno-naïve (such as transplant patients, AIDS sufferers or the foetus) can result in severe morbidity and mortality [9]. Similarly, reactivation from latency is a major cause of disease in certain transplant patients and late stage HIV patients suffering from AIDS [9– 11]. Consequently, understanding the regulation of viral gene expression during lytic infection and the mechanisms that regulate latency and reactivation, in vivo, is of paramount importance for future clinical intervention. 2. Infection of different cell types in vitro shows that the state of cell differentiation is an important determinant of permissiveness for infection During primary infection or reactivation in vivo, HCMV undergoes productive infection in a number of different cell types including smooth muscle, endothelial, epithelial, fibroblast, macrophage and dendritic cells [12]. Consistent with this, many of the cell types are also able to be infected in vitro — although the level of infection varies from cell type to cell type and is very dependent on virus isolates. For instance,

laboratory adapted strains of HCMV which have been routinely grown in primary human fibroblasts are unable to grow in myeloid, endothelial or epithelial cells due to mutation or loss of one or more of the viral UL128– UL131A genes. In contrast, clinical isolates of virus which have been grown in fibroblast cells for only a limited time efficiently infect differentiated myeloid and endothelial cells [13–19]. Intriguingly, cells of the early myeloid lineage such as bone marrow progenitor cells or monocytes do not support viral replication and this appears to be due to a differentiation-specific block in viral IE gene expression [20–24]; virus is taken up by the cell and viral genomes are transported to the nucleus but viral IE genes are not expressed. As these IE gene products are crucial for the subsequent expression of the viral early and late genes, this differentiationdependent block in IE gene expression essentially prevents lytic infection and likely underpins the mechanism by which these cell types act as one site of latent infection in vivo [25,26]. Consistent with this, monocytes isolated from healthy HCMV carriers do not express viral major IE RNAs but can reactivate major IE gene expression and produce infectious virus if they are differentiated to macrophages [27,28]. Similarly, naturally latently infected CD34+ progenitor cells, in which no IE mRNAs are detectable, also reactivate major IE gene expression and produce infectious virus after terminal differentiation to dendritic cells (DCs) ex vivo [29]. This ability of terminally differentiated macrophages and DCs to support reactivation of naturally latent virus is also consistent with the known permissiveness of terminally differentiated myeloid cells for experimental infection [20–24,30–33].

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It is clear, then, that whether the outcome of an HCMV infection is lytic or latent and whether cells are able to reactivate latent virus appears to depend on the ability of the infected cell to support robust viral IE gene expression. At least for cells of the myeloid lineage, this appears to be intrinsically linked to myeloid cell differentiation. 3. Post-translational modifications of histones around the HCMV MIEP mediates differentiation-dependent regulation of major IE gene expression HCMV major IE gene expression is under the control of the major IE promoter/enhancer (MIEP) (Fig. 1B): one of the most powerful transcriptional promoter/enhancers known [34,35]. It comprises an enhancer region (made up of an array of 17, 18, 19 and 21 bp repeat elements) and an upstream element (termed the modulator) which includes an imperfect dyad symmetry [35–37]. On the basis that the modulator region showed changes in DNAse I hypersensitivity upon differentiation of cells to a phenotype permissive for HCMV infection, it was long suggested that changes in binding of cellular transcription factors to the MIEP may play an important role in determining the permissiveness of cells for HCMV IE expression [36,38–41]. A number of analyses of the MIEP have been carried out in cell types which are either able or unable to support major IE expression to determine what cell factors differentially regulate IE gene expression. Initial studies used the human teratocarcinoma cell line NT2D1, as well as the myelomonocytic cell line THP1. Both cell lines are non-permissive for HCMV infection due to a block in major IE gene expression but differentiation results in a cell phenotype which supports IE gene expression and productive infection [42–46]. Analyses of regions of the viral MIEP responsible for this differentiation-dependent regulation of major IE gene expression as well as investigations of MIEP activity in fibroblast cells have shown that the HCMV MIEP not only contains DNA sequences which bind cellular transcription factors able to activate the promoter, such as NF-κB, CREB/ATF and AP1 [35,47–50], but also contains transcription factor binding sites for potent repressors of transcription such as Ying Yang 1 (YY1) and Ets-2 repressor factor (ERF) [36,40,51–59]. These observations suggested that a balance in the levels of repressors and activators of the MIEP can determine whether a specific cell type is permissive or non-permissive for viral major IE gene expression. Intriguingly, some of these differentiation-dependent transcriptional repressors of the MIEP, including YY1 and ERF [58–60], are transcription factors known to mediate transcriptional repression by recruitment of cofactors involved in post-translational modification of histones [60–62]. Consistent with the belief that negative regulation of viral IE gene expression in undifferentiated cells may involve chromatin structure of the MIEP, chromatin immune precipitation (ChIP) assays on HCMV infected undifferentiated NT2D1 cells or THP1 cells showed that the viral MIEP is predominantly associated with silencing proteins such as heterochromatin protein 1 (HP1). In contrast, upon infection of differentiated NT2D1 and THP1 cells, the MIEP becomes associated with histone markers of transcriptional activation, in particular acetylated histone H4 (corresponding to tetra-acetylated H4 on amino acids 2–19), which is correlated with robust major IE expression in these cells [63–65] (Fig. 2). Consistent with this, prior treatment of undifferentiated cells with the histone deactylase inhibitor Trichostatin A (TSA) results in increased major IE gene expression upon infection in these normally non-permissive cells [63,66]. 4. Differentiation-dependent changes in post-translational modifications of histones around the MIEP regulate virus latency and reactivation Clearly, the differentiation state of the cell determines whether the MIEP becomes associated with transcriptionally active chromatin or whether it becomes silenced by recruitment of transcriptionally

repressive chromatin marks upon HCMV infection. Similarly, differentiation appears to play a key role in reactivation of virus from naturally latent myeloid cells isolated from HCMV seropositive donors where terminal differentiation of latently infected CD34+ progenitors or monocytes reactivates latent virus [29]. An obvious question, then, is whether chromatin-mediated transcriptional repression of IE gene expression is also involved in the suppression of the viral lytic transcription programme during natural latent infection in vivo. Our own recent work has now shown that differentiationdependent chromatin remodelling of the viral MIEP by posttranslational modification of histones is, indeed, involved in the control of latency and reactivation of HCMV in vivo [29]. ChIP analysis of the HCMV MIEP carried out directly on cells isolated from naturally infected healthy seropositive donors has shown that in latently infected CD34+ cells and monocytes, the MIEP is associated with HP1β but not acetylated histones. However, ex vivo differentiation of these latently infected cells to mature DCs, which induce virus IE gene expression, results in chromatin remodelling of the viral MIEP. Specifically, HP1 is lost and the histones bound to the MIEP become acetylated; consistent with transcriptional activation of viral gene expression. These changes in post-translational modifications of histones associated with the viral MIEP correlate precisely with reactivation of infectious virus from these terminally differentiated mature DCs [29]. Identical results have been observed in experimental models of HCMV latency [22] (Fig. 2). The changes in histone marks associated with the viral MIEP which occur upon ex vivo differentiation of CD34+ progenitors to mature DCs also appear to be linked with changes in expression of specific cellular proteins identified as playing a putative role in the differentiation-dependent regulation of the viral MIEP in vitro. For instance, while YY1 and ERF show little change in levels of expression upon cell differentiation, differentiation does result in the downregulation of histone deacetylase 1 (HDAC1) protein [60,67]; a known co-repressor involved in transcriptional repression mediated by both YY1 [61] and ERF [60]. However, the differentiation of CD34+ cells to DCs does not result in global activation of all cellular promoters. So what determines the differentiation-specific acetylation of histones associated with the viral MIEP and its ensuing transcriptional activity? It is unlikely that a differentiation-dependent reduction of repressors of the MIEP is, in itself, sufficient to activate IE gene expression. Instead, activation of the viral MIEP also requires the concomitant expression of positive regulators of the MIEP which are not present in undifferentiated cells [47]. These factors are likely to mediate their activation by recruitment of co-factors involved in histone acetylation. Thus, the combination of transcription factor binding sites present in the MIEP confers on it the ability to respond to the balance of positive and negative cellular transcription factors present in undifferentiated or differentiated myeloid cells [1]. This differentiation-dependent activation of the viral MIEP may be mimicking the positive regulation of inflammatory genes normally associated with DC maturation [68] and has led to a model of HCMV latency in vivo (Fig. 3). Clearly, the chromatin structure of HCMV major IE gene promoter plays a profound role in determining whether a cell is able to support lytic infection and likely underpins whether infection results in a productive infection or whether the viral genome is repressed and, hence, latent. Similarly, differentiation of myeloid cells carrying latent viral genomes, results in reactivation of lytic gene expression and the production of infectious virus which correlates with differentiationdependent changes in post-translational modifications of histones associated with the MIEP. Recent studies with murine cytomegalovirus (MCMV) have also suggested that, during latency in the kidney, the MCMV MIEP becomes transcriptionally inactive as a result of changes in histone modifications and recruitment of transcriptional silencing factors to the viral MIEP [69]. However, whether MCMV latency fully reflects latency observed in HCMV is questionable as recent evidence to

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Fig. 2. Differentiation-dependent regulation of the MIEP in cell lines and primary myeloid cells is mediated through post-translational modifications of histones. A) Experimental HCMV infection of undifferentiated THP1 cells or NT2D1 cells results in a lack of IE gene expression and is correlated to a repressive chromatin structure around the MIEP (as detected by the recruitment of HP1β). In contrast, infection of THP1 or NT2D1 cell differentiated with phorbol esters (e.g. PMA) or retinoic acid, respectively, results in cells permissive for IE gene expression and is correlated to a transcriptionally active chromatin structure around the MIEP (as detected by acetylated histone H4). B) Identical observations shown in (A) are observed upon experimental infection of primary CD34+ progenitors or monocytes. C) The MIEP of HCMV present in naturally latent CD34+ progenitors, which do not produce infectious virus upon co-culture with fibroblasts, is associated with histone marks of transcriptional repression (HP1β). When these cells are differentiated ex vivo to DCs, virus reactivation occurs and this is correlated to changes in the chromatin structure of the MIEP associated with transcriptional activation (acetylated histone H4).

suggests that the MCMV immediate early 1 gene (IE1) is not required for establishment of latency or for reactivation, at least in the lungs [70,71]. 5. Promoters of latency-associated genes are also regulated by histone post-translational modifications Latency of HCMV in undifferentiated cells of the myeloid lineage appears to be enforced by the association of the viral MIEP with a repressive chromatin structure which prevents the undifferentiated cell from supporting robust viral IE gene expression. However, this repression clearly cannot extend to viral genes which are known to be expressed during viral latency. Although the identity and function of latency-associated genes are far from clear [1,26,72], a number of viral genes have been shown to be expressed during experimental latency but, more importantly, also during latent infection of myeloid cells in vivo [73–76]. One of these is the latency-associated transcript termed LUNA [73]. First identified by Bego and St Jeor, LUNA RNA is expressed from the complementary strand between HCMV UL81 and UL82 coding region. This RNA can be

detected in peripheral blood monocytes of healthy HCMV seropositive carriers and is believed to encode a small protein (approximately 14 kDa) of, as yet, unknown function: our own work has also confirmed expression of LUNA RNA in CD34+ progenitor cells of healthy carriers (Reeves and Sinclair, unpublished observations). The putative LUNA promoter [73] has all the characteristics of a myeloid specific promoter with multiple GATA factor binding sites and is activated by GATA-2 in vitro. In contrast to the viral MIEP, which is associated with histone marks of transcriptional repression in undifferentiated cells , the LUNA promoter is predominantly associated with acetylated histones in CD34+ cells after experimental infection (Reeves and Sinclair, unpublished). Clearly, the differences between the transcription factors binding sites in the MIEP and the LUNA promoter, coupled with the nuclear milieu of undifferentiated myeloid cells, results in the association of the MIEP with repressive chromatin but an association of the LUNA promoter with a transcriptionally active chromatin structure. This repression of IE gene expression prevents the lytic transcription programme and results in latent carriage of the viral genome with the concomitant expression of latency-associated genes such as LUNA.

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Fig. 3. A model of the regulation of HCMV latency and reactivation in vivo by chromatin structure of the MIEP. Following primary infection (A), HCMV establishes a latent infection of the CD34+ bone marrow mononuclear cells (B). The viral genome persists in the CD34+ cells in the absence of viral lytic gene expression due to the action of cellular transcriptional repressors which bind to the MIEP (such as ERF and YY1 — detailed in the text). These repressors recruit enzymes (e.g. ERF can interact directly with HDAC1 — see text) that modify chromatin around the MIEP such that it remains in a transcriptionally repressed state e.g. via recruitment of HP1β (C). However, if the CD34+ cells are differentiated to a mature DC phenotype by myeloid maturation signals and/or inflammatory signals, the reactivation of lytic gene expression results in the release of infectious virus progeny (D). The reactivation of lytic gene expression is concomitant with recruitment of transcriptional activators (e.g. NF-κB, AP1, CREB) and chromatin remodelling of the MIEP into a transcriptionally active state where the MIEP becomes associated with e.g. acetylated H4 through the action of, so far, unidentified histone acetyl transferases (HATs) (E). Chromatin remodelling of the MIEP allows the reactivation of viral lytic gene expression to occur following specific differentiation of the CD34+ cells to mature DCs. Modified with permission from [68].

The regulation of latency and reactivation of HCMV by chromatin structure of lytic promoters appears to be a common theme in the herpesviridae family. Work from a number of laboratories has shown that herpesvirus viral lytic gene expression, in general, is profoundly regulated by histone modification and that changes in the chromatinisation of the promoters regulating the major lytic genes of both EBV and HSV1 are also involved in the control of reactivation of these herpesviruses [71,77–81]. A number of observations from experiments analysing productive infection in fibroblast cell lines have suggested that post-translational modifications of histones also play a role in the regulation of HCMV gene expression during lytic infection. As discussed below, chromatin structure of the HCMV genome does indeed play a pivotal role in determining the initial efficiency of productive infection in normally differentiated permissive cells as well as in the temporal regulation of HCMV gene expression throughout the time course of the viral lytic life cycle. 6. The earliest events of HCMV lytic infection are regulated by histone post-translational modification Perhaps some of the earliest observations which suggested that chromatin structure of the viral genome is important for cytomegalovirus productive infection came from observations with MCMV which showed that TSA increased murine IE1 protein expression and virus production in MCMV infected cells [82]. These authors suggested that this phenomenon reflected intrinsic repression of MCMV IE gene expression mediated by histone deacetylases targeted to the viral genome by its localisation to nuclear structures known as nuclear domain-10 (ND10) bodies — nuclear structures advocated to play an important role during the earliest events of herpesvirus infection. Immediately post-infection, herpesvirus genomes become closely associated with ND10 bodies which appear to act as intrinsic anti-viral environments for herpesvirus gene expression [83–85]. Interestingly,

a number of major components of ND10 bodies have been shown to interact with chromatin modification enzyme activities such as histone deacetylases [86,87]. Thus, the increased MCMV IE gene expression in response to TSA observed by Tang and Maul was likely due to histone deacetylase inhibition [82] and suggested that the earliest events of MCMV lytic infection was subject to transcriptional regulation by histone modification. Consistent with this, it was also observed that Valproic acid, a pan-specific inhibitor of histone deacetylases, increased levels of HCMV IE and late protein expression in infected fibroblasts and epithelial cells [88–90]. More recently, our own studies and those of other laboratories have directly shown that ND10 components such as hDaxx, PML, and the hDaxx-associated protein ATRX are able to repress HCMV IE gene expression during lytic infection of permissive cell types [91–97]. At least in the case of hDaxx, this repression of the incoming HCMV genome is mediated through modifications of histones associated with the MIEP [97]. Specifically, over expression of hDaxx, in permissive cells normally capable of undergoing a full productive infection with HCMV, results in a marked decrease of IE gene expression. Similarly, knock down of endogenous hDaxx prior to infection results in increased IE gene expression that is clearly correlated with a loss of repressive chromatin structure around the MIEP [97]. Consistent with observations with MCMV infection, MIEPmediated repression could also be overcome by pre-treatment of cells with TSA [97]. This intrinsic chromatin-mediated repression of the MIEP appears to be an extremely rapid event. Within 3 h of infection the HCMV MIEP becomes a target for repressive chromatin which is, at least in part, mediated by hDaxx [97]. As predicted, inhibition of histone deacetylase activities by TSA prior to infection leads to immediate hyperacetylation of histones bound to the MIEP, and increases viral major IE gene expression, and advancement of the infection cycle by inducing premature viral early and late gene expression and DNA replication

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[98]. Consequently, it is possible that this initial intrinsic repression of IE gene expression that occurs upon HCMV infection of fully permissive cells is one of a number of mechanisms which impart on HCMV its relatively slow infection cycle. Interestingly, this “pre-immediate early” repression of the MIEP in fully permissive cells appears to be very vulnerable to the multiplicity of infection as high multiplicities of infection can effectively overcome the effects of over-expressed hDaxx [97]. Whether this is due to the ability of incoming virion proteins to target ND10 or whether increased copies of incoming viral genome are able to titrate out the MIEP repressive functions of the cell is, as yet, unclear [98]. Similarly, whether other components of ND10 such as PML and ATRX [92,95] also mediate their effects through chromatin structure of the MIEP, awaits further studies.

7. Chromatinisation of the HCMV gene promoters also regulates viral lytic gene expression throughout productive infection Other studies have suggested that histone modification also regulates HCMV gene expression throughout the course of lytic infection. For instance, activation of gene expression by IE72 and IE86, which act synergistically to drive early and late gene expression [99], appear to mediate activation of viral early promoters by sequestering histone deacetylases such as HDAC1, 2 and 3 to ensure high levels of acetylation of bound histones [100,101]. Consistent with this, there is now increasing evidence that IE72 and IE86 physically interact with these HDACs as well as the histone methyl transferases G9a and Suvar (3–9)H1 which are all important for post-translational modification of histones [100–102]. More direct evidence for the role of changes in histone modifications in the regulation of HCMV gene promoters throughout the course of productive infection has come from a recent analysis by Cuevas-Bennett and Shenk who have analysed changes in histone modifications on IE promoters as well as early and late promoters during the course of lytic infection [103]. In this study they observed dynamic acetylation and methylation patterns of histones associated with all classes of viral promoters. At IE times of infection early and late promoters are associated with histones carrying post-translational modifications known to be associated with transcriptional repression. As infection proceeds, early promoters and then late promoters gradually become associated with histone marks associated with transcriptional activation (acetylated H4 as well as acetylated H3K9 and H3K14). These transcriptionally active or repressed chromatin marks correlate exactly with the known temporal regulation of these promoters during the lytic infection cycle [103]. Our own work [98] is entirely consistent with these observations and also shows that, before increases in acetylated histones occur, early and late viral promoters are predominantly associated with dimethylated lysine 9 on histone H3 (di-methyl H3K9). These marks are indicative of the lack of transcription from these promoters at these respective times of infection and are characteristic of facultative heterochromatin [104]. This suggests that these promoters are in a chromatin state capable of rapidly becoming transcriptionally active, perhaps in response to activators such as IE72 and IE86 (Fig. 4). It is well established that, as herpesvirus lytic cycle proceeds, viral IE gene expression is negatively regulated at later times of infection, presumably as requirements for IE functions wane. At later times of HCMV infection, viral major IE gene expression is negatively autoregulated by binding of IE86 to the cis-repression sequence (crs) present in the MIEP [105–108]. We and others have shown that this IE86-mediated repression of the MIEP involves modification of histones associated with it. IE86 binds to the crs and recruits HDAC1 and histone methyltransferases such as G9a and Suvar(3–9)H1 forcing the MIEP into a repressive chromatin structure [103,109]. Interestingly, at this late time of infection, recruitment of HP1β to the

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MIEP is also evident which is indicative of tri-methyl H3K9 [109] and a more terminally repressed chromatin state [104]. More recently, it has been shown that IE86-mediated hypoacetylation of the MIEP chromatin may also occur very early in infection, prior to viral DNA replication when IE86 levels are building up [103]. Whether this has any direct functional consequences on viral replication is, as yet unclear. However, it probably reflects the functions of IE86 at IE times that are distinct from its activity as a negative auto-regulator at late times of infection. For instance, at IE/early times of infection, IE86 targets early and late viral promoters as well as cellular promoters for activation. This activation is likely due to IE86 sequestering or recruiting, histone modification enzymes [100–103,109]. Dynamic changes in the post-translational modifications of histones associated with all classes of viral promoters appear to occur during the course of productive infection (see Fig. 4). As the viral genome enters the nucleus, histones are recruited to the MIEP, forming a repressive chromatin structure [97,98]. At high multiplicities of infection, this initial repression may be overcome by delivery of high amounts of viral structural proteins or viral DNA [91– 94,97,110–112] which results in immediate association of the MIEP with marks of active chromatin [103]. Assembly of repressive chromatin also occurs at early and late viral promoters. However, once IE gene expression builds up, the modification of histones associated with early and late promoters is altered to marks associated with transcriptional activity. IE86 and its ability to interact with histone deacetylases and histone acetyltransferases [100– 103,109], likely play a crucial role in this. At late stages of infection, chromatin-mediated modulation of viral IE gene expression once again occurs. However, at this point of infection, binding of IE86 to the MIEP through the crs and its ability to recruit chromatin modifying enzymes mediates auto-repression (Fig. 4). Most of the observations discussed above, reflect changes at promoter sequences of IE, early and late genes. However, it is also clear that, as HCMV infection progresses, non-promoter regions of viral genes also become hyper-acetylated [103]. The role of this acetylation is unclear at present. It may be transcriptionally significant, or alternatively, may be involved in other processes such as viral DNA replication as these global increases in acetylated histones at viral promoter and non-promoter sites appear to require viral DNA replication [103]. The immediate response of the infected cell to incoming viral DNA, which results in repression of the incoming viral genomes by imparting on them a transcriptionally repressive chromatin structure, could reflect an intrinsic anti-viral host cell response to infection. Alternatively, it is possible that HCMV, as an obligate parasite, exploits an important cellular mechanism of transcriptional control, namely chromatin structure, to fulfil its requirement for a regulated cascade of expression of its own genome. Whichever is the case, much data now suggest that chromatin remodelling plays an important role in the regulation of all classes of HCMV gene expression throughout the course of lytic infection. 8. Chromatin assembly during lytic infection occurs on a substantial proportion of viral DNA which must be removed before packaging into virions It is now becoming increasingly clear that histones are deposited on viral genomes during both latent and lytic infection and that the post-translational modification of these chromatin proteins appears to play a profound role in control of viral gene expression. Whether this association of histones with viral DNA results in bona fide nucleosomal structures has been addressed in recent work from Nitzsche et al. [113] which suggests that the observed association between viral genome and histones during productive infection does, indeed, reflect specific chromatin-like assembly on viral DNA. These authors systematically analysed histone occupancy on the HCMV genome during lytic infection and observed significant deposition of histones on

292 J. Sinclair / Biochimica et Biophysica Acta 1799 (2010) 286–295 Fig. 4. Chromatin structure of promoters of all classes of HCMV genes is regulated by histone modifications throughout the time course of lytic infection. At low multiplicities of infection, non-nucleosomal incoming viral genomes are subject to intrinsic repression of the MIEP by cellular factors such as those present in ND10 (red arrows) resulting in H3K9 methylation and recruitment of HP-1β. This “pre-immediate early” repression is transiently overcome by incoming pp71 tegument protein likely allowing some IE72 expression which is, in turn, able to disrupt ND10 (Box 1). This results in robust activation of the MIEP and its association with histone marks indicative of transcriptional activation such as acetylated H4 as well as acetylated H3K9 and H3K14. At the same time, histones associated with early and late promoters are in a repressed chromatin state associated with methylated H3K9 (Box 2). As IE72 and IE86 build up, these activators allow de-repression of early promoters, probably by interaction with histone deacetylases (HDAC1–3). Concomitantly, the build up of IE86 initiates repression of the MIEP by binding to the crs and recruiting HDAC1 and histone methyltransferases (HMT) such as G9a and Suvar(3–9)H1(Box 3). At late times of infection, late gene expression is activated which correlates with the association of late promoters with histone marks indicative of transcriptional activation such as acetylated H4 as well as acetylated H3K9 and H3K14. Major IE expression is then robustly repressed by histone deacetylation with HDAC1, histone methylation with G9a and Suvar(3–9)H1 and recruitment of repressor proteins such as HP1β (Box 4).

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viral DNA which had many characteristics of nucleosomes. However, the average histone level on the HCMV genome was lower than for cellular chromatin, suggesting that at early stages of infection viral chromatin may be in a more irregular state [113]. They also observed that histone occupancy on viral genomes increased substantially during infection in a DNA replication-dependent manner [113]. Others, however, have not observed major changes in the levels of histones interacting with viral DNA during the time course of infection [103]. Perhaps confounding these observations, is the fact that they result from analyses of viral genomes which will only give an average of the histone occupancy or the histone acetylation state of viral genomes. If the assembly of chromatin on these incoming viral genomes is relatively inefficient (for instance, replication-independent chromatin assembly is less efficient than replication-dependent chromatin assembly [114]), only a very small proportion of incoming viral genomes might become associated with chromatin and this might be further compounded at very high multiplicities of infection. Under these conditions, many of the analyses described above could be difficult to interpret. However, our recent observations [98] have shown that, at low multiplicities of infection (0.1 pfu/cell), as much as 20% of incoming HCMV genomes are associated with the histone H3 at IE times of infection. That these genomes are the functionally important ones is supported by the observation that at 8 h post-infection only approximately 10% of viral MIEPs are associated with active RNA Pol II (perhaps suggesting that a substantial number of incoming viral genomes are defective or unable to initiate major IE transcription). Consequently, the changes in histone post-translational modifications that have been observed during HCMV lytic infection, especially at low multiplicities of infection, are likely to occur on a significant and transcriptionally important population of incoming viral genomes. The importance of multiplicity of infection and the particle or viral DNA/pfu ratio in these types of assays is also underscored by the fact that pre-treatment of cells with TSA or Valproic acid, prior to infection, increased IE gene expression only at low multiplicities of infection [88–90,97]. The observation that incoming HCMV genomes appear to be extensively associated with histones upon lytic infection and this extensive histone deposition also occurs on replicated viral DNA [103,113] begs the question of how histones are removed prior to genome encapsidation as HCMV particles do not contain histones [115]. It is likely that an organised chromatin disassembly programme must accompany packaging of HCMV DNA [113]. Where and how this occurs is unclear, but it is likely that it will involve the use of cellular and perhaps viral re-modelling factors. Although it is still unclear as to whether the assembly of chromatin on incoming HCMV genomes results in histone–DNA complexes indicative of true cellular chromatin, HCMV genomes are extensively associated with cellular histones which regulate their transcription. Why this appears to be different from other herpesviruses such as HSV-1, studies on which have shown a predominantly non-nucleosomal structure of viral DNA in lytically infected cells (see [81] for review), is unclear. However, many recent analyses are beginning to suggest that these original observations may need to be reviewed in the light of multiplicity of infection or the potent effect of incoming virion transactivators on chromatin structure-associated transcriptional activation (Triezenberg, this volume). 9. Does the intrinsic repression of HCMV IE gene expression observed upon lytic infection of normally permissive cells also underpin the mechanism which defines latency? Infection of terminally differentiated fibroblast cells in vitro normally results in potent IE gene expression and full productive infection. However, this infection cycle appears to be subject to attempts by the cell to suppress transcription of incoming viral genomes mediated, at least in part, through nuclear domains such as

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ND10. A valid question is whether these types of mechanisms are the same ones which “force” latency on incoming viral genomes. Our own observations [116], in contrast to the results obtained by Saffert and Kalejta [117], suggest that removal of these repressors of viral gene expression has no effect on IE expression in cells which show a differentiation-dependent repression of the MIEP through histone post-translational modification: hDaxx knockdown does not prevent repression of the incoming viral genomes [116]. The reason for the differences observed in these two studies is, as yet, unclear. Our own view is that latency of HCMV is fundamentally associated with the differentiation state of the cell. In undifferentiated cells, such as CD34+ progenitor cells, the balance of positive/negative regulators of the MIEP results in repression of the MIEP that is mediated by binding of high levels of differentiation-dependent repressive transcription factors. These factors are capable of recruiting families of co-repressor proteins which modify histone on the MIEP resulting in an overall repressive chromatin state. Once cell differentiation occurs, the nuclear milieu changes to an overall environment conducive to MIEP activity but the MIEP becomes subject to the intrinsic anti-viral environment of the nucleus (i.e. reactivation of MIEP activity in differentiated cells could be considered to be akin to lytic infection at very low multiplicities of infection and in the absence of any virion transactivators [68]). Whether this intrinsic repression is overcome by a steady build up of e.g. IE72 protein, which is capable of targeting and disrupting ND10-mediated repression or whether other viral functions associated with latent infection are able to help overcome this intrinsic repression, (see McGregor Dallas and Sinclair, Abstr. 33rd Int Herpesvirus Workshop), awaits further studies. 10. Conclusions The expression of HCMV major IE expression is crucial for driving virus productive infection. Therefore the mechanisms that regulate MIEP activity not only underpin the temporal control of the viral lytic transcription programme but also control the potent repression of the MIEP observed during latent infection and reactivation of latent virus. A number of studies have begun to address the regulation of HCMV viral gene expression through chromatin structure in experimental or natural latent infection [22,29,63–65] as well as, during lytic infection [97,103,109]. Generally, these studies agree that post-translational modifications of histone associated with HCMV gene promoters are crucial for the regulation of viral gene expression during latency and reactivation but also during the lytic cycle. Whether the histone/DNA complexes observed on viral genomes are absolutely identical to cellular chromatin may be missing the point. Regardless, these complexes undoubtedly play a potent role in the regulation of viral gene expression in ways akin to the regulation of cellular genes. This is, perhaps, not surprising considering the dependence of herpesviruses on the cellular transcription machinery. As the sophistication in studying chromatin biology increases, our understanding of the role of chromatin in the viral life cycle, from viral gene expression through to viral DNA replication and viral DNA packaging, will also undoubtedly deepen. Fully understanding the ways these cell-derived transcriptional control mechanisms regulate herpesvirus gene expression will be crucial for a thorough understanding of viral latency and reactivation as well as understanding intrinsic cellular anti-viral defence mechanisms. All of these may, one day, be exploited for novel anti-viral strategies. Acknowledgements I would like to thank all members, past and present, whose work has contributed to the studies described here. I also apologise to colleagues in the field whose work could not be cited due to space

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