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. Author manuscript; available in PMC: 2012 Nov 25.
Published in final edited form as: Virology. 2011 Sep 22;420(2):73–81. doi: 10.1016/j.virol.2011.08.019

Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX

Bryan C Mounce 1, Fei Chin Tsan 1, Lindsay Droit 3, Sarah Kohler 2, Lisa A Cirillo 2, Vera L Tarakanova 1,*
PMCID: PMC3204369  NIHMSID: NIHMS328048  PMID: 21943826

Abstract

Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

Keywords: gammaherpesvirus, DNA damage response, H2AX, gammaherpesvirus protein kinase

Introduction

Gammaherpesviruses, such as human Epstein-Barr virus (EBV) and Kaposi’s Sarcoma associated herpesvirus (KSHV), are ubiquitous pathogens that establish a life-long infection and are associated with a wide range of malignancies (Damania, 2004; Jung et al., 1999; Tarakanova et al., 2005; Young and Rickinson, 2004). All mammalian herpesviruses, including gammaherpesviruses, encode a protein kinase, illustrating the importance of this viral function in herpesvirus infection. Herpesvirus protein kinases share common biological characteristics, including association with virions, nuclear localization, and support of viral replication (Gershburg et al., 2007; Moffat et al., 1998; Krosky et al., 2003; Wolf et al., 2001; Chang et al., 2009; Gershburg and Pagano, 2008). Furthermore, kinases encoded by herpes simplex virus (HSV) and mouse gammaherpesvirus 68 (MHV68) facilitate viral latency in vivo (Asano et al., 2000; Tarakanova et al., 2010). Because both lytic and latent viral life cycles are likely to contribute to gammaherpesvirus-induced tumorigenesis, development of specific inhibitors of gammaherpesvirus kinases is an attractive therapeutic approach.

A detailed understanding of the mechanism by which gammaherpesvirus kinases facilitate viral replication and latency is critical for the development of effective kinase inhibitors. Gammaherpesvirus protein kinases affect multiple processes in infected cells, including expression of select viral genes, viral DNA synthesis, and nuclear egress of viral capsids (Feederle et al., 2009; Gershburg et al., 2007; Lee et al., 2008; Meng et al., 2010). Lamin A/C phosphorylation by EBV kinase BGLF4 likely contributes to nuclear egress (Lee et al., 2008; Meng et al., 2010); however, the mechanism by which gammaherpesvirus kinases regulate viral gene expression and DNA synthesis is not known. Furthermore, most of the phenotypes of gammaherpesvirus kinases have been examined in transformed latently-infected cell lines; thus, the functions of these protein kinases in primary physiologically-relevant cell types are largely not understood. Exquisite species specificity of EBV and KSHV further restricts studies of physiologically relevant functions of human gammaherpesvirus kinases.

MHV68 is genetically and biologically related to human EBV and KSHV and is associated with induction of lymphoproliferative disease, including B cell lymphomas (Efstathiou et al., 1990; Virgin et al., 1997; Tarakanova et al., 2005; Tarakanova et al., 2008). MHV68 represents a tractable experimental system that supports studies of de novo gammaherpesvirus lytic infection in primary, physiologically-relevant cell types and allows genetic manipulation of both host and virus. MHV68-encoded protein kinase orf36 enhances viral replication in primary macrophages (Tarakanova et al., 2007). Furthermore, both MHV68 orf36 and a related EBV protein kinase BGLF4 induce DNA damage response (DDR), independent of the presence of viral DNA (Tarakanova et al., 2007). DDR is a cellular tumor suppressor network that functions to detect and repair DNA lesions within the host genome. DDR is manipulated by several viruses to facilitate viral replication [reviewed in (Weitzman et al., 2010)]. Specifically, histone variant H2AX and Ataxia-Telangiectasia mutated (ATM) kinase, critically important members of the proximal signaling events within the DDR network, are both required for efficient MHV68 replication in primary macrophages (Tarakanova et al., 2007). Importantly, the mechanism by which H2AX or ATM facilitates MHV68 replication is not known.

In this study we report that orf36 facilitated transcription of MHV68 Replication and Transcription Activator (RTA) and replication of viral DNA during de novo lytic infection of primary macrophages. While the effects of orf36 on RTA transcription were restricted to low multiplicity of infection (MOI) conditions, the effects of orf36 expression and enzymatic activity on viral DNA synthesis were MOI-independent. Furthermore, attenuated RTA transcription and MHV68 DNA synthesis were found in H2AX-deficient primary macrophages, indicating that the host DNA damage response contributes to the stimulation of MHV68 replication by orf36.

Results

Orf36 facilitates RTA transcription

Because gammaherpesvirus kinases are virion components (Johannsen et al., 2004), these kinases are likely to affect several early steps of infection, such as expression of RTA, an immediate early gammaherpesvirus protein that is essential for lytic replication and reactivation from latency (Liu et al., 2000; Lukac et al., 1998; Sun et al., 1998; Wu et al., 2000; Wu et al., 2001). To determine whether orf36 expression or its enzymatic activity had an effect on RTA transcription early in infection, primary bone marrow-derived macrophages were infected with wild type MHV68, N36S mutant unable to express orf36 due to a translational stop within the orf36 gene, or 36KN mutant that expresses an enzymatically inactive protein due to a point mutation within the catalytic domain (Hwang et al., 2009; Tarakanova et al., 2007). RTA transcript levels were measured by quantitative RT-PCR and normalized to GAPDH transcript.

A single cycle of MHV68 replication takes 40–48 hours in primary macrophages infected at 1 PFU/cell, with RTA gene expression first detected between 12 and 16h post infection (data not shown). At 16h post infection, under conditions of low multiplicity of infection (MOI, 1 PFU/cell), the levels of RTA transcript were decreased 3–4-fold in macrophages infected with either orf36 mutant as compared to the wild type virus (Fig. 1A, p<0.05 for N36S and 36KN compared to wild type virus). The observed difference in RTA transcript was not due to decreased infectivity of orf36 virus mutants, as levels of cell-associated viral DNA were similar in all experimental groups (Fig. 1B). Levels of RTA transcript were attenuated as late as 30h post infection in macrophages infected with the N36S and 36KN mutants (5-fold, Fig. 1C), indicating that the effects of orf36 on RTA transcription were not due to a kinetic delay. Interestingly, infection at an MOI of 10 PFU/cell rescued the attenuation of RTA transcription in N36S- and 36KN-infected macrophages (Fig. 1D). Thus, orf36 expression and enzymatic activity facilitated RTA transcription in primary macrophages in an MOI-dependent manner.

Figure 1. Orf36 facilitates transcription of RTA.

Figure 1

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Primary bone marrow derived macrophages were infected at 1 (A, B, C) or 10 (D) PFU/cell. RNA was isolated at 16 (A, D) or 30 hours post infection (C), and levels of RTA transcript measured by quantitative RT-PCR with subsequent normalization to the corresponding GAPDH levels. B. Total DNA was isolated at 16 h post infection, MHV68 DNA measured by real time PCR, and normalized to corresponding cellular DNA. Data in every panel were pooled from 3–7 independent experiments. * − p<0.05 compared to wild type.

Transcription of an RTA-dependent gene is attenuated in the absence of orf36

RTA directly activates its own promoter and the promoter of orf57, an early viral gene encoding an important regulator of viral and host transcription and RNA stability (Pavlova et al., 2005; Majerciak and Zheng, 2009). Furthermore, overexpression of RTA in the context of lytic replication results in transcriptional upregulation of a majority of MHV68 lytic genes (Hair et al., 2007), suggesting that the effects of RTA on viral gene expression are likely driven by a plethora of mechanisms. To determine whether attenuation of RTA transcription in the absence of orf36 had an impact on the expression of an early MHV68 gene directly induced by RTA, orf57 transcript levels were measured in primary macrophages infected with wild type MHV68 or orf36 mutant viruses. The levels of orf57 transcript were decreased 3- to 5-fold at 16h post infection in macrophages infected with either N36S or 36KN mutant (Fig. 2A, p<0.05), indicating that the attenuation of RTA expression in the absence of orf36 was sufficient to decrease transcription of a downstream RTA-dependent viral gene. The levels of orf57 transcript were similarly attenuated in macrophages infected with N36S and 36KN mutants at 1 PFU/cell (Fig. 2A, p>0.05). Attenuated orf57 transcript levels were rescued under high MOI conditions (Fig. 2B), consistent with orf36-independent transcription of RTA in macrophages infected at a high MOI (Fig. 1D). Similarly, transcript levels for orf72, an additional RTA-responsive gene (Allen, III et al., 2007), were decreased in the absence of orf36 or its catalytic activity under low MOI conditions (Fig. 2C)

Figure 2. Expression of orf57, but not ssDBP, is attenuated in the absence of functional orf36.

Figure 2

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Primary bone marrow derived macrophages were infected at 1 (A, C, E) or 10 (B) PFU/cell. RNA was isolated at 16 h post infection and levels of orf57 (A, B), orf72 (C) or ssDBP (E) transcript measured by quantitative RT-PCR with subsequent normalization to corresponding GAPDH levels. Data were pooled from 3–4 independent experiments. D. Cell lysates were collected at indicated times post infection and subjected to western analysis using an antibody specific for ssDBP or cellular beta actin. * − p<0.05 compared to wild type. NS – not significant, p>0.05 compared to wild type.

We next examined whether the observed effects of orf36 deficiency on orf57 and orf72 transcription were representative of specific or global regulation of viral gene expression. MHV68 orf6 encodes a conserved single-stranded DNA binding protein (ssDBP) necessary for viral DNA replication. A single transcript encoding ssDBP is produced early in the lytic cycle (Kapadia et al., 1999). While ssDBP transcript is upregulated in an RTA-overexpressing MHV68 mutant (Hair et al., 2007), the orf6 promoter has not been identified; thus, it is not clear whether RTA can directly induce orf6 transcription. To determine whether orf36-dependent changes in RTA transcription affected ssDBP expression, levels of ssDBP protein were measured in primary macrophages using a polyclonal antibody generated against recombinant His-tagged protein containing amino acids 1–435 of MHV68 ssDBP. Immune serum detected a protein of approximately 125 kDa in lysates from infected, but not mock-infected macrophages and mouse embryonic fibroblasts, consistent with the predicted molecular weight of MHV68 ssDBP (Fig. 2D, data not shown). ssDBP levels were similar in the presence and absence of functional orf36 in infected primary macrophages under both high and low MOI conditions (Fig. 2D). Furthermore, orf36 had no effect on the levels of ssDBP transcript at 16 h post infection (Fig. 2E). Thus, effects of orf36 no MHV68 gene expression were limited to RTA and viral genes known to be directly regulated by RTA.

H2AX facilitates RTA transcription

Orf36 expression and enzymatic activity is necessary and sufficient to induce DDR, as evidenced by an increase in serine 139- phosphorylated H2AX (γH2AX) and activation of ATM in MHV68-infected or orf36-expressing cells (Tarakanova et al., 2007). Importantly, orf36 may directly phosphorylate serine 139 of H2AX, providing a possible mechanism by which this kinase induces DDR in infected cells (Tarakanova et al., 2007). H2AX is an H2A variant that constitutes 2–25% of all H2A incorporated into nucleosomes (Rogakou et al., 1998). γH2AX rapidly accumulates at DNA breaks and serves as an adaptor protein that facilitates recruitment and retention of downstream DDR and DNA repair proteins at the DNA lesion (Stucki and Jackson, 2006).

Because orf36 facilitated RTA transcription at a low MOI, and because of the kinase-dependent DDR induction in infected macrophages, we wanted to determine whether H2AX expression affected RTA transcription. Primary macrophages were derived from bone marrow of H2AX deficient or wild type littermates and infected with wild type MHV68 or the orf36 mutant viruses. RTA transcription was decreased approximately 3-fold in H2AX-deficient macrophages infected at a low MOI with wild type MHV68 as compared to the wild type MHV68 infection of wild type cells (Fig. 3A, p<0.05), indicating that H2AX expression was important for optimal RTA transcription. As expected, the levels of RTA transcript were decreased in H2AX wild type macrophages infected with the orf36 mutants (Fig. 3A). Interestingly, RTA transcript levels were further attenuated in H2AX-deficient macrophages infected with the orf36 mutant viruses (Fig. 3A; p<0. 05 for either mutant). Because orf36 is required for induction of serine 139 phosphorylated H2AX (γH2AX) in infected cells (Tarakanova et al., 2007), these data suggested that other, serine 139 phosphorylation-independent functions of H2AX are important for RTA transcription. Interestingly, similar to the MOI-dependence of the orf36 phenotype, the RTA transcript levels were comparable in H2AX-deficient and wild type cells infected at a high MOI with wild type MHV68 (Fig. 3B).

Figure 3. H2AX facilitates RTA transcription.

Figure 3

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A, B. Primary bone marrow de rived macrophages were isolated from H2AX-deficient mice or wild type littermates and infected as indicated. RNA was isolated at 16 h post infection and levels of RTA transcript measured by quantitative RT-PCR with subsequent normalization to corresponding GAPDH levels. C, D, E. Primary bone marrow derived macrophages were infected with the indicated viruses at an MOI of 1, and chromatin subjected to immunoprecipitation at 16h post infection using anti-H2AX (C), anti- γH2AX (D, E), or an irrelevant antibody (IgG) (C, D, E). Fold enrichment of indicated DNA sequences was calculated using ΔΔCT method. Pooled data from 2–5 independent experiments are shown in each panel. F. Schematic representation of the core and distal RTA promoters. Arrows indicate positions of primers used to amplify sequences outside of the core and distal RTA promoters.

The effects of H2AX on RTA transcription could occur in cis and/or in trans, via H2AX-dependent changes in the cell environment or via direct recruitment of DDR proteins to the RTA promoter. In the latter scenario, γH2AX would be expected to associate with the known RTA promoters (Gray et al., 2008; Liu et al., 2000). Because association of MHV68 DNA with core histones has not been characterized in the context of lytic infection, we first wanted to establish whether H2AX-containing nucleosomes associated with MHV68 DNA. Primary macrophages were infected at an MOI of 1 with wild type MHV68 or either orf36 mutant virus, and the association of total H2AX with the core RTA promoter was measured by chromatin immunoprecipitation (ChIP) at 16h post infection. H2AX was associated with the core RTA promoter under all three experimental conditions, albeit the levels were somewhat decreased in 36KN-infected cells (Fig. 3C). Next, the association of γH2AX with both core and distal RTA promoters was assessed by ChIP. In the context of physiological DDR, γH2AX domains can extend as far as 1 Mbp from the double stranded DNA break (Savic et al., 2009). To determine if γH2AX associated with regions beyond the RTA promoters, we examined sequences 5′ and 3′ of the core and distal RTA promoter for γH2AX association (schematic in Fig. 3F). The levels of γH2AX associated with core RTA promoters were at or below IgG background in macrophages infected with N36S or 36KN virus mutants (Fig. 3E, white bars; data not shown). Interestingly, elevated levels of γH2AX were associated with the core RTA promoter in wild type MHV68-infected macrophages (Fig. 3E), indicating that the expression of enzymatically-active orf36 induced γH2AX at the core RTA promoter, a marker that is likely to recruit downstream DDR signaling components. γH2AX enrichment extended beyond the immediate core promoter, as sequences upstream and downstream of the core promoter were also immunoprecipitated with the anti-γH2AX antibody in wild type- but not 36KN-infected macrophages (Fig. 3E). When distal RTA promoter sequences were probed by ChIP, γH2AX was found to associate with the distal promoter and proximal sequences in wild type- but not 36KN-infected cells (Fig. 3D). Interestingly, the overall levels of γH2AX associated with the distal RTA promoter were lower compared to those found at the core promoter, suggesting that orf36 enzymatic activity was required to differentially regulate γH2AX enrichment at the RTA promoters. Thus, expression of enzymatically active orf36 induced γH2AX within and in proximity to the core and distal RTA promoters and H2AX expression facilitated transcription of RTA in an MOI-dependent manner.

Orf36 and H2AX facilitate MHV68 DNA synthesis

While the effects of orf36 expression on RTA transcription were MOI-dependent, orf36 stimulates MHV68 replication in primary macrophages in an MOI-independent manner (Tarakanova et al., 2007). Because viral DNA synthesis is a step of the replication cycle that is downstream of immediate early and early gene expression, effects of orf36 on viral DNA accumulation were measured under both high and low MOI conditions. As shown in Fig. 4A, B, functional orf36 was required for efficient MHV68 DNA accumulation in primary macrophages at either MOI. Because of the effects of orf36 on RTA and orf57 expression, transcription of six MHV68 genes required for viral DNA synthesis was measured at 30h post infection, shortly after initiation of the viral DNA synthesis. Neither orf36 expression nor its enzymatic activity was required for efficient transcription of MHV68 DNA synthesis genes under all conditions tested (Fig. 4C, D, data not shown). Furthermore, protein levels of ssDBP, one of the six viral genes required for DNA synthesis were similar in wild type and orf36-mutant-infected macrophages at 30h post infection (Fig. 2D). Thus, orf36 facilitated MHV68 DNA synthesis in an MOI-independent manner. Importantly, the observed defect in viral DNA accumulation in orf36-deficient infections could not be attributed to decreased expression of viral DNA synthesis machinery.

Figure 4. Orf36 facilitates MHV68 DNA synthesis.

Figure 4

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Primary bone marrow derived macrophages were infected at 10 (A, C) or 1 (B, D) PFU/cell. A, B. Total DNA was isolated at indicated times post infection. MHV68 DNA was measured by real time PCR and normalized to corresponding cellular DNA. C, D. RNA was isolated at 30 hours post infection and levels of the indicated MHV68 DNA synthesis gene transcripts measured by quantitative RT-PCR with subsequent normalization to the corresponding GAPDH levels. Data in every panel were pooled from 2–5 independent experiments.

To determine whether H2AX expression enhanced MHV68 DNA replication, primary macrophages were isolated from H2AX wild type or deficient mice, infected with wild type MHV68, and viral DNA measured at several times post infection. A modest decrease (2–3-fold) in MHV68 DNA levels was observed at 48h post infection in H2AX deficient macrophages infected at a high MOI (Fig. 5A), consistent with similar viral titers produced in H2AX deficient and wild type macrophages under high MOI conditions (Tarakanova et al., 2007). Importantly, MHV68 DNA accumulation was significantly decreased at 48 h post infection in H2AX deficient macrophages infected at a low MOI (Fig. 5B), suggesting that the expression of H2AX was important for efficient MHV68 DNA replication under conditions of low virus inoculum. Cell-associated viral DNA was not significantly different (p<0.05) at 0 hpi, suggesting that this difference was not due to reduced infectivity in H2AX deficient cells.

Figure 5. H2AX expression facilitates viral DNA synthesis in an MOI-dependent manner.

Figure 5

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Primary bone marrow derived macrophages were isolated from H2AX deficient-mice or wild type littermates and infected as indicated. Total DNA was isolated and MHV68 DNA measured by real time PCR, with subsequent normalization to corresponding cellular DNA. Data in every panel were pooled from 2–3 independent experiments.

Discussion

Working model

Based on the results of our study, we would like to propose a working model of the mechanism by which orf36 regulates MHV68 lytic replication in primary macrophages (Fig. 6). Orf36 targets several steps of the lytic cycle. As a virion component, orf36 stimulates immediate early RTA transcription via orf36 ability to induce DDR with subsequent induction of γH2AX at the RTA core promoter. Importantly, other orf36 functions, such as modulation of type I IFN signaling, may also contribute to regulation of RTA transcription. Because the role of MHV68 RTA in viral DNA synthesis is not clear, attenuated RTA transcription under low MOI conditions may or may not affect downstream viral DNA synthesis in orf36- or H2AX-deficient cells. However, the ability of orf36 to stimulate viral DNA synthesis at high MOI likely stems from DDR- and RTA-independent functions of this viral kinase.

Figure 6. Working model.

Figure 6

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Orf36 regulates both immediate early gene expression and viral DNA synthesis in primary macrophages. Regulation of RTA expression is evident at a low MOI and depends, in part, on the ability of orf36 to induce DDR and H2AX phosphorylation in infected cells. Orf36 facilitates viral DNA synthesis in an MOI-independent manner. The regulation of viral DNA synthesis is likely to involve other, DDR-independent functions of orf36.

Regulation of RTA transcription by orf36 and H2AX

Attenuated expression of several lytic gammaherpesvirus genes in the absence of gammaherpesvirus protein kinase was previously demonstrated in EBV-infected cell lines induced to reactivate from latency (Feederle et al., 2009; Gershburg et al., 2007). Furthermore, abundance of several late MHV68 proteins is decreased in macrophages infected with orf36 mutants, in part, due to decreased viral DNA synthesis at late time points post infection(Tarakanova et al., 2007). Our study is the first to demonstrate the effect of gammaherpesvirus protein kinase on immediate early viral lytic gene expression, a finding generated using de novo lytic gammaherpesvirus infection of primary cells. While latency is the predominant gammaherpesvirus life cycle in the immunocompetent host, infection of naïve cells occurs during acute and chronic infection and is important for the maintenance of the pool of infected cells in chronically infected host, as demonstrated for both MHV68 and EBV in vivo (Liang et al., 2009; Hoshino et al., 2009). Thus, an understanding of early events of de novo gammaherpesvirus infection is highly relevant for the development of efficient therapeutic control of both acute and chronic phases of gammaherpesvirus infection.

Our studies show that regulation of immediate early gene expression by orf36 is specific to certain MHV68 lytic genes, such asRTA, orf57, and orf72. Interestingly, all three genes are directly transactivated by RTA (Pavlova et al., 2005; Majerciak and Zheng, 2009; Allen, III et al., 2007). It is tempting to speculate that orf36 selectively enhances expression of lytic MHV68 genes directly regulated by RTA. In the KSHV system, 19 RTA binding sites were identified within the viral genome (Chen et al., 2009), with many of RTA binding sites localized within the viral gene promoters. A comprehensive analysis of MHV68 genes directly regulated by RTA has not been performed.

One mechanism by which orf36 facilitates RTA transcription is via induction of DDR, as demonstrated by the association of γH2AX with the RTA promoters in wild type- but not orf36 mutant-infected macrophages and requirement of H2AX expression for efficient RTA transcription (Fig. 3). While many genes are transcriptionally induced during DNA damage response in an ATM-dependent manner (Bredemeyer et al., 2008), transcription of genes proximal to the double stranded DNA break is thought to be silenced by ATM and RNF8/RNF168, a complex of E3 ubiquitin ligases(Shanbhag et al., 2010). Importantly, both RNF8 and RNF168 are targeted for degradation in HSV-infected cells (Lilley et al., 2011; Lilley et al., 2010), indicating that herpesviruses have the potential to counteract-associated DDR transcriptional silencing. In the future it will be important to determine whether RTA promoter-associated γH2AX facilitates recruitment of other DDR proteins, including A TM and RNF8/RNF168, and effects of such recruitment on RTA transcription. In addition to direct effects on viral promoters, H2AX an d, likely, other DDR proteins, may facilitate RTA transcription indirectly, via effects on other cellular pathways, such as MAPK signaling that is activated during DDR and is known to induce RTA and orf57 promoter activity (Li et al., 2010; Pan et al., 2006).

Importantly, orf36-mediated regulation of viral gene expression is likely to extend beyond its ability to manipulate DDR. Orf36 and a related EBV kinase BGLF4 inhibit IFNβ transcription and signaling in infected cells(Hwang et al., 2009; Wang et al., 2009). While type II IFN is known to inhibit RTA transcription(Goodwin et al., 2010), future studies should determine whether type I IFN signaling regulates RTA promoter activity and whether orf36-dependent inhibition of type I IFN plays a role in RTA expression. Orf36’s effects on RTA transcription were evident under low, but not high MOI, suggesting that other viral tegument protein(s) may compensate for the absence of orf36 by stimulating RTA transcription via alternative, DDR-independent mechanism.

Regulation of viral DNA synthesis by orf36 and H2AX

While orf36 facilitated MHV68 DNA synthesis independent of MOI, effects of H2AX expression on the accumulation of viral DNA were limited to low MOI, suggesting that additional functions of orf36, such as inhibition of type I IFN signaling may be required for MHV68 DNA synthesis at a high MOI. Multiple DDR proteins are known to colocalize with the nuclear replication compartments of EBV(Kudoh et al., 2005; Kudoh et al., 2009); however, the role of these proteins in viral DNA synthesis in primary cells is not clear. In this study we show that H2AX, an important component of the proximal DDR signaling, has a greater role in MHV68 DNA synthesis at a low, but not high MOI, suggesting that either DDR is no longer needed to enhance viral DNA replication following a large inoculum or that other DDR proteins compensate in the absence of H2AX. Future studies will discriminate between the two possibilities.

Importantly, attenuation of MHV68 DNA synthesis in the absence of a functional orf36 could not be attributed to the lack of viral DNA synthesis machinery, as transcription of all six MHV68 genes required for viral DNA synthesis was orf36-independent. EBV protein kinase BGLF4 contributes to the efficient viral DNA synthesis(Gershburg et al., 2007) and is known to phosphorylate several EBV DNA synthesis proteins (Zhu et al., 2009); however, it is not clear whether this phosphorylation affects the activity of EBV DNA synthesis machinery. Future studies should determine whether, in addition to potential effects on the activity of viral DNA synthesis machinery, gammaherpesvirus kinases affect the assembly and localization of viral replication compartments, as gammaherpesvirus kinases have the potential to modify nuclear architecture via phosphorylation of nuclear lamins (Lee et al., 2008; Meng et al., 2010). The attenuated transcription of orf72 in the absence of orf36 (Fig. 2C) is an intriguing finding, as UL97, a betaherpesvirus protein kinase induces G1/S progression via hyperphosphorylation of pRb (Hume et al., 2008). Orf72-encoded v-cyclin is required to induce pRb phosphorylation during lytic MHV68 infection (Upton et al., 2005). Thus, orf36, via stimulation of orf72 transcription, may contribute to cell cycle regulation in the context of MHV68 infection.

In conclusion, our studies highlight several of what undoubtedly is a plethora of yet undiscovered mechanisms by which gammaherpesviruses usurp the host DDR network throughout the lytic replication. This virus-host interaction has important implications for the pathogenesis of gammaherpesvirus under DDR insufficient conditions such as those observed in all malignant cells, as well as in human diseases associated with mutations of the DDR genes.

Materials and Methods

Animals and primary cell cultures

C57BL/6J (BL6) mice were obtained from Jackson Laboratories (Bar Harbor, Maine), H2AX-deficient mice were a kind gift of Dr. Fred Alt (HHMI, Harvard Medical School). All mice were housed and bred in a specific-pathogen-free barrier facility in accordance with federal and institutional guidelines. All experimental manipulations of mice were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. Bone marrow was harvested from mice between 7 and 10 weeks of age. Primary bone marrow macrophages were generated and infected as previously described (Tarakanova et al., 2007).

Western blot analysis

Macrophages were collected into laemmli buffer and analyzed as previously described (Lenschow et al., 2005). Antibodies used were anti-ssDBP (1:1000 dilution) anti-β actin (1:2500 dilution, Novus Biologicals, Littleton, CO), and a secondary goat anti-mouse or anti-rabbit HRP -conjugated secondary antibody (Jackson Immunoresearch, Westgrove, PA). Anti-ssDBP antibody was raised in rabbits immunized with purified 6X-His-tagged antigen corresponding to residues1–4351 of the MHV68 ssDBP protein sequence (Cocalico Biologicals, Reamstown, PA).

qRT-PCR quantitation of viral messages

Total RNA was harvested, DNAse treated, and reverse transcribed as described in (Tarakanova et al., 2009). cDNA was serially diluted (8-fold), cDNA dilutions and corresponding minus RT reactions assessed, in triplicate, by real time PCR using iCycler (Bio-Rad, Hercules, CA). MHV68 gene-specific cDNA was amplified using primers decribed in (Tarakanova et al., 2009). Orf72-specific cDNA was amplified using the following primers: Forward: 5′-GAC-GGA-GGT-CTT-TGC-ACA-CAC-AAA-3′; Reverse: 5′-ATC-GCA-GCG-AAA-GAG-AAC-ACG-ATG-3′. Delta Ct method was used to quantify relative abundance of each cDNA, using corresponding GAPDH levels for normalization. Ct values of minus RT controls did not exceed background levels.

Viral DNA measurement

Total DNA was isolated from infected cells by washing cells twice with PBS and scraping into 300 μL lysis buffer (0.75% SDS, 40 μg/ml proteinase K, 10 mM Tris-HCl pH 8.1, 1 mM EDTA). Samples were incubated overnight at 56°C and DNA extracted twice with phenol/chloroform and once with chloroform. DNA was precipitated with 30 μL 3M NaOAc and 1.2 mL EtOH and resuspended in TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA). Viral DNA was quantified using ΔCT method using gene 50 and GAPDH primers described in (Tarakanova et al., 2009).

Chromatin Immunoprecipitation

Infected cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, and cross linking was quenched with 125mM glycine. Cells were washed three times with cold PBS, collected in 3 mL PBS by scraping, and pelleted at 4°C. Cell pellet was resuspended in 2 mL lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with Halt protease cocktail (Thermo Scientific, Rockford, IL) and sonicated to produce approximately 500-bp fragments (Cell Disruptor 185, Branson Sonifier, Danbury, CT). Sonicated samples were cleared of debris by centrifugation and portion of supernatant was set aside for analysis of input DNA. Chromatin was precleared with 80 μL protein G sepharose beads (Invitrogen, Carlsbad, CA) for 1h at 4°C, immunoprecipitated with 2 μg of anti-H2AX (Bethyl Laboratories, Montgomery, TX), anti-γH2AX (Millipore, Billerica, MA), or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) overnight, and incubated with 60 μL protein G for 2h at 4°C. Immunoprecipitates were washed at 4°C with buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl pH 8.1) containing low salt (150 mM NaCl) and high salt (500 mM NaCl). The samples were subsequently washed with LiCl buffer (0.25% LiCl, 1% IGEPAL-CA630, 1% sodium deoxycholate, 2 mM EDTA, 10 mM Tris-HCl pH 8.1) and twice with PBS. Chromatin was eluted twice by incubating immunoprecipitates with elution buffer (100 mM NaHCO3, 1% SDS, 10 mM DTT) at room temperature for 15 minutes. Cross links of immunoprecipitated and input samples were reversed by treatment with NaCl overnight at 65°C. Proteins were cleared by proteinase K treatment at 45°C for 1h, and DNA was purified using GeneJET PCR purification kit (Fermentas, Glen Burnie, MD). Gene-specific DNA was amplified utilizing GAPDH primers described in (Tarakanova et al., 2009), core RTA promoter primers (Forward: 5′-TTT-AGC-ATC-TGC-CCG-ACC-TGA-GA-3′; Reverse: 5′-AAT-GGA-CCT-TGA-AAC-CCG-TGA-AGG-3′), 5′ core RTA (Forward: 5′-AAG-CTG-GTG-AGG-CTG-GGA-AGT-TAT-3′; Reverse: 5′-TGT-TAT-GTG-CCT-GCC-CTT-CTC-ACT-3′), 3′ core RTA (Forward: 5′-ATG-TCC-CTT-CAA-AGG-CTG-AGG-AGA-3′; Reverse: 5′-TCG-CTG-CAG-AAA-TTC-CCT-CGT-AGT-3′), 5′ distal RTA (Forward: 5′-TTC-ACT-TTC-ACC-TGG-GCC-ACA-TTG-3′; Reverse: 5′-AAT-CCT-GGA-ACT-TCC-TGT-CCT-CCA-3′), distal RTA (Forward: 5′-AGG-TGG-TGT-TGG-GTT-AGT-ACA-GCA-3′; Reverse: 5′-TAG-TGA-CAG-GTA-AAG-CAT-AGC-CTG-GG-3′), and 3′ distal RTA (Forward: 5′-CCA-ACT-ATG-ATT-CTG-CCC-AAG-GAC-CA-3′; Reverse: 5′-TGA-TCA-GGA-ATT-CTA-CAC-AGA-GGC-3′) and enrichment was calculated using the ΔΔCT method.

Highlights.

  • Gammaherpesvirus protein kinase facilitates immediate early gene expression

  • H2AX, a critical DNA damage response protein, contributes to viral gene expression

  • Gammaherpesvirus protein kinase is required for efficient viral DNA synthesis

Acknowledgments

We are grateful to Frederick Alt for his generous gift of H2AX deficient mice and Herbert (Skip) Virgin for kindly sharing an anti-ssDBP antibody. Expert technical and managerial support was provided by Brittani Wood. We also thank Amy Hudson’s, William Jackson’s, and Scott Terhune’s laboratories for helpful discussions.

This work was supported by F32DK079649-01 (S.K.), R01DK073641 (L.A.C), Advancing Healthier Wisconsin and 1R56AI084889 (V.L.T.). The funding sources had no involvement in the published studies.

Footnotes

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