Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 1

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1 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 1 George Miller,*,{ Ayman El Guindy, z Jill Countryman, z Jianjiang Ye, z and Lyn Gradoville* *Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520; { Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520; z Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut I. Two Life Cycles of EBV and KSHV: Latency and Lytic Replication II. Virally Encoded Lytic Cycle Activator Genes A. Upstream and Downstream Events in Lytic Cycle Activation B. Agents That Induce the Lytic Cycle C. Role of Phosphorylation in the Downstream Functions of the EBV ZEBRA Protein III. Conclusions: Some Unsolved Mysteries About Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses References The seminal experiments of George and Eva Klein helped to define the two life cycles of Epstein Barr Virus (EBV), namely latency and lytic or productive infection. Their laboratories described latent nuclear antigens expressed during latency and discovered several chemicals that activated the viral lytic cycle. The mechanism of the switch between latency and the lytic cycle of EBV and Kaposi s sarcoma associated herpesvirus (KSHV) can be studied in cultured B cell lines. Lytic cycle activation of EBV is controlled by two viral transcription factors, ZEBRA and Rta. The homologue of Rta encoded in ORF50 is the lytic cycle activator of KSHV. Control of the lytic cycle can be divided into two distinct phases. Upstream events control expression of the virally encoded lytic cycle activator genes. Downstream events represent tasks carried out by the viral proteins in driving expression of lytic cycle genes and lytic viral DNA replication. In this chapter, we report three recent groups of experiments relating to upstream and downstream events. Azacytidine (AzaC) is a DNA methyltransferase inhibitor whose lytic cycle activation capacity was discovered by G. Klein and coworkers. We find that AzaC rapidly activates the EBV lytic cycle but does not detectably alter DNA methylation or histone acetylation on the promoters of the EBV lytic cycle activator genes. AzaC probably acts via a novel, yet to be elucidated, mechanism. The lytic cycle of both 1 Nobel Symposium on Cancer Research, Molecular Oncology from Bench to Bedside, in Honor of George and Eva Klein. Advances in CANCER RESEARCH X/07 $35.00 Copyright 2007, Elsevier Inc. All rights reserved. DOI: /S X(06)

2 82 George Miller et al. EBV and KSHV can be activated by sodium butyrate (NaB), a histone deacetylase inhibitor whose activity in disrupting latency was also discovered by G. Klein and coworkers. Activation of EBV by NaB requires protein synthesis; activation of KSHV is independent of protein synthesis. Thus, NaB works by a different pathway on the two closely related viruses. ZEBRA, the major downstream mediator of EBV lytic cycle activation is both a transcription activator and an essential replication protein. We show that phosphorylation of ZEBRA at its casein kinase 2 (CK2) site separates these two functions. Phosphorylation by CK2 is required for ZEBRA to activate lytic replication but not to induce expression of early lytic cycle genes. We discuss a number of unsolved mysteries about lytic cycle activation which should provide fertile territory for future research. # 2007 Elsevier Inc. I. TWO LIFE CYCLES OF EBV AND KSHV: LATENCY AND LYTIC REPLICATION From the earliest days of research on Epstein Barr virus (EBV) it was clear that not all cells that were infected with the virus produced mature viral progeny. When the Henles originally described the viral capsid antigen, using indirect immunofluorescence techniques, they observed the antigen only in a subpopulation of cells from Burkitt s lymphoma (Henle and Henle, 1966). Single cell cloning experiments showed that the progeny of single cells invariably gave rise to populations of cells in which only a subpopulation expressed the viral capsid antigen (Miller et al., 1970). This result suggested that all cells contained latent viral genomes which were periodically reactivated to produce virus in a subpopulation. Moreover, some cell lines, such as the famous Raji Burkitt s lymphoma cells, never spontaneously expressed viral capsid antigen (Epstein et al., 1966). However, Raji cells could be demonstrated to contain an antigen that was recognized by sera from EBV infected individuals. Complement fixation was required to detect this antigen (Armstrong et al., 1966; Pope et al., 1969). In a classic paper in 1973, Beverly Reedman and George Klein used immunofluorescence to detect the complement fixing antigen in the nucleus (Reedman and Klein, 1973)(Fig. 1A). This nuclear antigen was found in all cells in cell lines that spontaneously produced virus in a few cells and in those, such as Raji, that did not produce virus. Further work showed that Raji did not express capsid proteins because EBV DNA in Raji is unable to replicate in a lytic fashion. The EBV genome in Raji cells contains a deletion of BALF2, the gene encoding single stranded DNA binding protein, required for lytic EBV DNA replication (Decaussin et al., 1995; Zhang et al., 1988). The complement fixing nuclear antigen discovered by Reedman and Klein, which they called EBNA (EB nuclear antigen), is now known to consist of the products of at least six different EBV genes. The component of EBNA most commonly detected by infected individuals is EBNA1, the product of the EBV BKRF1 gene (Fischer et al., 1984; Summers et al., 1982). EBNA1 is responsible for partitioning of EBV DNA during latency from parental to daughter cells and for mediating

3 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 83 Fig. 1 Detection of latent and lytic EBV gene expression in B95 8 cells (Miller and Lipman, 1973). (A) Anticomplement immunofluorescence according to the method of Reedman and Klein (Reedman and Klein, 1973). The majority of cells show nuclear staining. Two bright cells (arrows) contain viral capsid antigen. (B) Fluorescent in situ hybridization (FISH) with EBV Bam H1 W as a probe. Latently infected cells contain approximately 20 genomes per cell. Following treatment with TPA approximately 25% of the cell population contain brightly staining viral replication factories indicative of lytic cycle activation. attachment of the viral genome to cell chromosomes (Kapoor et al., 2001; Yates et al., 1984). During latency, the EBV genome exists as an extrachromosomal element (Nonoyama and Pagano, 1972). Most latently infected cells contain approximately 20 copies of viral DNA that can be visualized by fluorescence in situ hybridization (Fig 1B). When the virus is induced to replicate, either spontaneously or after the addition of a chemical inducing agent, the viral DNA in a subpopulation of cells aggregates into replication factories (Gradoville

4 84 George Miller et al. et al., 2002). Thus, there are two distinct life cycles of EBV, latency and lytic replication, which can be studied in cultured lymphoid cells. The relationship between EBVand lymphoid cells offers a unique experimental system in which to study control of the latency to lytic cycle switch. Avery similar system exists for study of the related gammaherpesvirus, Kaposi s sarcoma associated herpesvirus (KSHV), which remains latent in cultured B cells derived from primary effusion lymphoma (Cesarman et al., 1995). A number of laboratories, including those of George and Eva Klein, have spent considerable effort over the past 40 years attempting to unravel the complex biology of the latency to lytic cycle switch of the oncogenic human gammaherpesviruses. Aside from the obvious advantage, by comparison to other herpesviruses, that the latency to lytic cycle switch of gammaherpesviruses can be experimentally manipulated in cultured cells, why all this effort? It is a biologically interesting example of the combinatorial regulation of eukaryotic gene expression. Control is exerted by groups of virally and cellular encoded transcription factors, acting both as activators and repressors. Epigenetic controls of promoter chromatinization and methylation come into play. The latency to lytic switch has obvious implications for the pathogenesis of malignant and nonmalignant diseases associated with EBV and KSHV. While latency may be the predominant life cycle form in virus associated cancers, the virus must replicate and assume a lytic state in order to be transmitted between cells and among individuals. Being able to manipulate the transition between latency and the lytic cycle offers the promise of translational application to virus associated cancers. For example, tumor lysis might be achieved by activating lytic viral replication. Thereafter, spread of virus to new cells could be inhibited by antiviral drugs directed at viral DNA replication (Westphal et al., 2000). Although the latency to lytic cycle switch can be manipulated in cell culture, particularly by the addition of one or more chemical inducing agents, little is understood about the physiologic stimulus that triggers the event in vivo. The stimulus may be external, for example receipt of an activating cytokine signal or removal of an inhibitory signal. Or the physiologic stimulus may be internal, reflecting the metabolic state of the cell, or its position in the cell cycle. II. VIRALLY ENCODED LYTIC CYCLE ACTIVATOR GENES Through study of a defective EBV genome in a Burkitt s lymphoma cell line, P3J HR 1, Jill Countryman and colleagues discovered that the lytic cycle of EBV was controlled by a transcription factor protein encoded in the EBV BZLF1 gene (Countryman and Miller, 1985). When expressed in latently

5 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 85 infected cells, this protein, variably named ZEBRA, Zta, or EB 1, is sufficient to activate the entire EBV lytic cycle cascade, leading to production of infectious virus (Grogan et al., 1987). ZEBRA protein, in some ways analogous to the prokaryotic lambda phage repressor, was the first example of latency to lytic cycle switch protein encoded for by a eukaryotic virus. ZEBRA plays many roles in the lytic phase of the viral life cycle. ZEBRA is essential to activate lytic cycle genes (Feederle et al., 2000). One of its earliest and most critical functions is to activate expression of another transcription factor, Rta, encoded by the viral BRLF1 gene (Kolman et al., 1996). ZEBRA and Rta then act in synergy to activate transcription of a subset of early lytic cycle genes many of which encode proteins required for lytic viral DNA replication (Quinlivan et al., 1993; Ragoczy and Miller, 1999). Rta appears to be capable of activating a distinct set of viral genes independent of ZEBRA (Chen et al., 2005; Ragoczy and Miller, 1999). ZEBRA represses the action of Rta on some promoters in a temporally controlled manner (El Guindy and Miller, 2004; Ragoczy and Miller, 1999). ZEBRA plays a distinct role in activating viral lytic DNA replication by binding to the origin of lytic cycle replication (orilyt) and by interacting with and recruiting viral proteins that are essential for lytic replication (Baumann et al., 1999; Fixman et al., 1992; Gao et al., 1998; Lieberman et al., 1990; Schepers et al., 1993, 1996) (Fig. 10B). Experiments using EBV bacmids in which the BZLF1 and BRLF1 have been insertionally inactivated show that both genes are required for lytic cycle activation (Feederle et al., 2000). An unanswered question is why EBV employs two different viral transcription factors to control the lytic cycle. KSHV or Human herpesvirus 8 encodes homologues of ZEBRA and Rta (Fig. 2A). K8 bzip, the ZEBRA homologue, exists in several spliced isoforms (Lin et al., 1999). K8 bzip is essential for viral DNA replication, but unlike ZEBRA, it does not seem to play a role as a transcriptional activator of early genes (Fig 2B). The job of activation of KSHV viral early lytic cycle genes is performed by KSHV ORF50 protein, the homologue of EBV Rta (Sun et al., 1998). A. Upstream and Downstream Events in Lytic Cycle Activation Early experiments with EBV showed that expression of ZEBRA strictly correlated with lytic cycle activation (Fig. 3A). When ZEBRA was constitutively expressed, as the result of genome rearrangements in the defective EBV genome (so called het DNA), or induced to be expressed, as the result of treatment of cells with inducing agents such as phorbol esters or sodium butyrate, the virus was forced to enter the lytic cycle. When neither ZEBRA protein nor BZLF1 mrna were expressed, the virus was in latency. Expression of ZEBRA obligated the virus to enter the full lytic cascade.

6 86 George Miller et al. Fig. 2 Lytic cycle activator genes encoded by EBV and KSHV. (A) Homologous genes and gene products of EBV and KSHV. (B) Functions of the activator genes in lytic cycle expression. Notably K8 bzip, the ZEBRA homologue in KSHV, is a replication protein but not an activator of early gene transcription. Thus, the latency to lytic cycle switch might be envisioned to be composed of two distinct sets of events: (1) upstream events leading to expression of the lytic cycle activator genes (ZEBRA and Rta in the case of EBV; ORF50 in the case of KSHV) and (2) downstream events by which the products of the lytic cycle activator genes control viral and cellular gene expression and viral and cellular DNA replication. A simple model for the entry pathway into the EBV lytic cascade (Fig. 3B) suggests that during latency the BZLF1 and BRLF1 genes are repressed. The mechanism of repression is likely to involve both epigenetic mechanisms such as chromatization and DNA methylation of the BZLF1 promoter, Zp, and BRLF1 promoter, Rp, as well as occupancy of these promoters by specific repressors. One candidate repressor for Zp, called ZEB1, has been identified (Kraus et al., 2001, 2003). Following application of an inducing stimulus, there is likely to be a signaling pathway that allows cell encoded activators to bind to Zp and Rp. Candidates for this phase include cellular proteins such as Sp1, c/ebp, AP 1, and CREB. These cellular factors lead to low level expression of ZEBRA and Rta, which then amplify the signal by autostimulating and cross stimulating each other s expression. ZEBRA specifically binds to several ZEBRA response sites in Rp. A methylated CpG is embedded in one of those sites. ZEBRA binds preferentially to methylated DNA (Bhende et al., 2004, 2005). Rta autostimulates Rp by an indirect mechanism that involves interaction with other proteins such as those of the Sp1 family (Ragoczy and Miller, 2001). ZEBRA appears to mediate all of its downstream activities by a direct DNA binding mechanism. We have completed saturation mutagenesis of

7 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 87 Fig. 3 Models for activation of the EBV lytic cycle cascade. (A) An early ZEBRA centric model in which all inducing stimuli lead to activation of ZEBRA expression and thereafter downstream events. (B) A later model incorporating control of the EBV lytic cycle by BRLF1 and BZLF1 genes. During latency, Rp and Zp, the promoters of these genes are repressed by histones (H). These promoters contain TPA response elements (TRE) and ZEBRA (Z) response elements (ZRE). Following induction, cell encoded activators (A) including Ap 1 factors bind to the promoters. In the autostimulation phase both ZEBRA (Z) and Rta (R) activate Rp. Autoactivation of the Rp by Rta is indirectly mediated by X, probably a member of the Sp1 family.

8 88 George Miller et al. the DNA recognition domain of ZEBRA and found that any mutation that eliminated ZEBRA binding to its highest affinity ZEBRA response elements (ZRE) abolishes the biologic activity of the protein (Heston et al., 2006). EBV Rta and its homologue KSHV ORF50 activate gene expression by more diverse mec hanisms than does ZEBR Fig. A ( 4A and B). EBV Rta has three types of target genes. One group consists of those genes it activates independently of ZEBRA by binding to Rta response elements in the promoter (Chen et al., 2005). A second group of genes is activated as the result of Rta and ZEBRA both binding to the promoter of responsive genes (Francis et al., 1999; Quinlivan et al., 1993). A third group of target genes is activated indirectly by Rta operating through binding sites for cellular transcription factors (Furnari et al., 1992). Rta may influence the activity of cellular Fig. 4 Mechanisms of action of the EBV Rta and KSHV ORF50 proteins. (A) EBV/Rta activates one group of genes autonomously by a direct DNA binding mechanism. A second group of genes is activated in synergy with ZEBRA. A third group of genes is activated via cellular transcription factors. (B) KSHV/ORF50 activates one group of genes by direct DNA binding and two other groups by interaction with cellular transcription factors.

9 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 89 transcription factors by protein protein interactions and by activating signaling cascades that lead to posttranslational modification of the cellular transcription factors (Adamson et al., 2000). KSHV ORF50 also activates downstream targets by a variety of mechanisms. These mechanisms can be distinguished by ORF50 mutants that do or do not bind to DNA (Chang et al., 2005). The promoters of direct targets, such as PAN and K12, share response elements that bind ORF50 protein (Chang et al., 2002). One group of indirect target genes has been shown to be activated by ORF50 mutants that do not bind DNA. These mutants interact with the RBP J protein (Liang et al., 2002). B. Agents That Induce the Lytic Cycle A number of different chemicals have been found to induce the EBV and KSHV lytic cycle (Fig. 5 presents a partial list). All the chemicals appear to operate by activating expression of the virally encoded lytic cycle activator genes, that is, they impinge on crucial upstream events. George Klein was a leader in investigating chemicals that could activate EBV lytic gene expression. For example, Klein and his colleagues, Janos Luka and Bengt Kallin, were the first to show that sodium butyrate, an inhibitor of histone deacetylase (HDAC), is a potent lytic cycle activator in some cell backgrounds (Luka et al., 1979). In our laboratory, we often use two prototype cancer cell lines to study the mechanism of lytic cycle activation HH514 16, an EBV infected cell line from Burkitt s lymphoma, and HH B2, a KSHVinfected cell line from primary effusion lymphoma (Gradoville et al., 2000). The advantage of these cell lines as model systems is that they manifest a very low background level of spontaneous lytic cycle activation; on application of the inducing stimulus a high proportion (up to 50%) of the cells undergo lytic Fig. 5 A partial list of agents commonly used to induce the EBV and KSHV lytic cycle in cultured lymphoid cell lines. Prototype lymphoma cell lines used to study gammaherpesvirus lytic cycle activation in our laboratory are HH from Burkitt s lymphoma and HH B2 from primary effusion lymphoma.

10 90 George Miller et al. cycle activation. In both of these cell lines, sodium butyrate reproducibly activates the viral lytic cycle. There are at least three major unanswered questions about the mechanism by which lytic cycle activation stimuli work. First, the list of chemicals in Fig. 5 shows that known inducing stimuli operate via many different modes of action. The inducing chemicals include activators of protein kinase C (PKC), inhibitors of HDACs, and DNA methyltransferase inhibitors. Is there a final common pathway to activation of the promoters of the lytic cycle activator genes or can several distinct unrelated events activate these genes? Second, even with the most potent activation stimulus in the most susceptible cell line, only a subpopulation of the cells respond. What accounts for this refractory subpopulation? Is it due to permanent or transient changes in the viral or cellular genome? Finally, the same inducing stimulus does not activate lytic cycle gene expression in all cells backgrounds. What accounts for the differences among cell lines in their response to inducing stimuli? 1. THE SAME STIMULUS DOES NOT ACTIVATE EBV LYTIC CYCLE GENE EXPRESSION IN ALL CELL BACKGROUNDS At the symposium George Klein pointed out that the differences in response of EBV containing cell lines to various inducing stimuli that work by many diverse mechanisms represents a major unsolved puzzle about the gammaherpes lytic cycle switches. For example, our laboratory stocks of B95 8 cells, a marmoset lymphoblastoid cell line derived by in vitro immortalization with EBV, respond strongly to phorbol ester (Fig. 6A). The cell line HH514 16, a subclone of the Burkitt s lymphoma Jijoye line, fails to express lytic cycle genes after treatment with phorbol ester (Gradoville et al., 2002). TPA is a potent agonist for PKC. The levels of PKC activity increase in both responsive and unresponsive cell lines following treatment with TPA. Therefore, it is likely that some additional event, downstream of PKC activation, that is required for EBV lytic activation does not take place in the HH Burkitt s lymphomaderived cell line. Conversely, EBV lytic cycle gene expression is reproducibly activated to a high level in HH cells by HDAC inhibitors such as sodium butyrate and Trichostatin A (TSA) (Fig. 6B). Activation of the lytic cycle in HH cells by HDAC inhibitors is not accompanied by detectable activation of PKC nor is lytic cycle activation by HDAC inhibitors blocked by bisindolymaleimide, a potent inhibitor of PKC (Gradoville et al., 2002). These results lead to the conclusion that activation of PKC is not universally required for EBV lytic cycle activation. B95 8 cells do not increase lytic cycle gene expression following treatment with sodium butyrate or TSA. However, as expected, these agents induce hyperacetylation of histone tails, H3 and H4, in B95 8 cells. The hyperacetylation is both global (i.e., detectable by increased levels of H3 and H4 hyperacetylation on immunoblots) and specific to the promoters of the BZLF1 (ZEBRA) and BRLF1 (Rta) genes (i.e., detectable by

11 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 91 A TPA Cell line A (e.g., B95-8) PKC TPA Cell line B (e.g., HH514-16) PKC B TSA Cell line A (e.g., B95-8) TSA ach3 and ach4 Cell line B (e.g., HH514-16) ach3 and ach4 Fig. 6 Inducing agents exhibit cell line specific behavior. (A) Effect of phorbol ester (TPA). TPA activates PKC in two EBV containing cell lines, but in only one cell line, A, is this stimulus adequate to induce the lytic cycle. Latent genomes: open black circles; replicating genomes: black shaded circles; virions: open gray circles. (B) Effect of an HDAC inhibitor, Trichostatin A (TSA). In both cell lines, TSA causes hyperacetylation of histone tails ach3 and ach4. Only in cell line B does this stimulus lead to EBV lytic cycle reactivation. chromatin immunoprecipitation with antibody to acetylated H3 and histone tails H4) (Countryman et al., 2006). These results show that histone hyperacetylation is neither a sufficient nor universal stimulus to activate EBV lytic gene expression. There appears to be an activity, in addition to hyperacetylation of Rp and Zp, that is essential for HDAC inhibitors to disrupt EBV latency. This activity may involve acetylation of nonhistone proteins or other properties of the HDAC inhibitors that are not related to protein acetylation. 2. MYSTERIES ABOUT THE MECHANISMS BY WHICH AZACYTIDINE INDUCES EBV LYTIC GENE EXPRESSIONS Samuel Ben Sasson and George Klein demonstrated that 5 Azacytidine activated EBV early lytic gene expression in human lymphoid lines latently infected with EBV (Ben Sasson and Klein, 1981). We have found that in some B cell backgrounds, such as HH cells, the DNA methyltransferase

12 92 George Miller et al. inhibitor 5 Aza 2 0 deoxcytidine (5Aza2 0 dc) very rapidly and potently induces expression of the EBV BRLF1 and BZLF1 mrnas. An increase in the levels of lytic cycle activator mrnas can be detected within 4 h after addition of 5Aza2 0 dc (Fig. 7A). The effects are more rapid than those of A HH HRS Induction: Rp (4.0) - NaB 5Aza2 dc NaB 5Aza2 dc NaB 5Aza2 dc NaB 5Aza2 dc Markers (Kb) 4.4 Rp (3.0) Zp (1.0) RNaseP (0.35) Probe: Z(300) B Fold stimulation Hours HH NaB 5Aza2 dc

13 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 93 C (a) Zp 359/ / / / / 78 % Methylation +13/ / /+189 Uninduced NaB 5Aza2 dc (b) Rp % Methylation / / / / / / 95 58/ 57 49/ 48 5/ 4 HH cells Uninduced NaB 5Aza2 dc Fig. 7 Activation of the EBV lytic cycle by 5 Aza 2 0 deoxycytidine (5Aza2 0 dc) and sodium butyrate (NaB). Experiments were conducted in HH cells (cell line B in Fig. 6). (A) Kinetics of activation of BRLF1 (Rp 3.0) and BZLF1 (Zp 1.0) mrnas by the two stimuli. (B) Hyperacetylation of Rp determined by chromatin immunoprecipitation with antibodies to acetylated H3. (C) Lack of effect of the two inducing stimuli on CpG methylation of Zp or Rp determined by bisulfite sequencing 8 h after induction.

14 94 George Miller et al. sodium butyrate and, at early times, relatively more pronounced on the BLZF1 mrna than on the BRLF1 mrna. 5Aza2 0 dc does not detectably effect the acetylation state of histone H3 on Rp at 4 or 6 h after addition (Fig. 7B). Moreover, at 8 h after addition neither Azacytidine (AzaC) nor sodium butyrate affects the methylation state of the promoters of the BZLF1 and BRLF1 genes (Fig. 7C). Thus, at times when 5Aza2 0 dc is inducing the lytic cycle, there is no detectable change either in DNA methylation of the promoters of the lytic cycle activator genes or in hyperacetylation of H3 on these promoters. It is unlikely, therefore, that EBV lytic cycle induction by 5Aza2 0 dc is operating directly by epigenetic alterations on the promoters of BZLF1 and BRLF1. There appears to be another yet to be discovered mechanism of action of AzaC. 3. DIFFERING EFFECTS OF CYCLOHEXIMIDE ON LYTIC CYCLE REACTIVATION OF EBV AND KSHV BY SODIUM BUTYRATE An important unanswered question is whether stimuli that induce the EBV and KSHV lytic cycle work on transcription factors that are already expressed in the cell at the time when the cell receives the lytic cycleinducing stimulus. The transcription factors may be poised, ready to work after they are activated, for example, by posttranslational modification. Alternatively, lytic cycle activation stimuli might change the epigenetic state of the promoters of the lytic cycle activation genes or remove repressors, thus allowing preexisting competent transcription factors access to the promoters. If either of these two scenarios is operative, a lytic cycle activation stimulus, such as sodium butyrate, might not require de novo protein synthesis. Rather, sodium butyrate might work by activating a signal transduction cascade that modifies a repressor or by opening chromatin via the hyperacetylation of histone tails. As originally defined by Roizman and colleagues, a herpesvirus immediateearly gene can be transcribed in the presence of an inhibitor of protein synthesis such as cycloheximide (CHX) (Frenkel et al., 1973; Honess and Roizman, 1974). An explanation for immediate early kinetics in herpes simplex virus is that the virus carries a preformed transcription factor (VP16) within the virion. This transcription factor activates immediate early downstream genes following de novo infection of cells with the virus. The question whether EBV BZLF1 and BRLF1 and KSHV ORF50 are immediate early genes has not yet been carefully investigated. It is unknown whether EBV BZLF1 and BRLF1 and KSHV ORF50, the lytic cycle activator genes, behave with immediate early kinetics on de novo infection or on lytic cycle reactivation after addition of a potent lytic cycle inducing stimulus such as sodium butyrate.

15 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 95 We recently investigated the question of immediate early behavior on lytic cycle reactivation in two prototypic cell lines infected with EBV or KSHV in which sodium butyrate activates the lytic cycle (Fig. 5). CHX was added at intervals after the inducing stimulus. In HH cells (Fig. 8), appearance of the EBV BRLF1 and BZLF1 mrnas was inhibited by addition of CHX added up to 4 h after sodium butyrate. By contrast, induction of ORF50 mrna by sodium butyrate in HH B2 cells was resistant to the action of CHX at all times. In fact, if CHX was added simultaneously with sodium butyrate, there was enhanced expression of ORF50 mrna, a finding consistent with the known activity of CHX in stabilizing mrna. These results indicated that for lytic cycle reactivation, de novo protein synthesis appeared to be required for expression of the EBV BRLF1 and BZLF1 mrnas but not for KSHV ORF50 mrna. One limitation of this initial experiment was that the effects of CHX on lytic cycle reactivation were studied in two different cell backgrounds. Many primary effusion lymphoma cell lines are dually infected with EBV and KSHV (Cesarman et al., 1995). In one such dually infected cell line, BC 1, sodium butyrate induces lytic cycle expression of both EBV and KSHV. Five hours after CHX was added together with sodium butyrate (NaB), Fig. 8 Differential effects of CHX on lytic cycle induction of EBV and KSHV by sodium butyrate (NaB). Each cell line was untreated or treated with sodium butyrate (NaB). CHX was added simultaneously with NaB or at 2 h intervals thereafter. Lytic cycle activator mrnas were analyzed on Northern blots 8 h after addition of NaB. (A) Probed for EBV BRLF1 (Rp) and BZLF1 (Zp) mrnas. (B) Probed for KSHV ORF50 mrna.

16 96 George Miller et al. the abundance of EBV BRLF1 mrna was 33% of the level measured after NaB in the absence of CHX (Fig. 9A). At the same time, in the same cell line, the expression of KSHV ORF50 mrna was not inhibited; in fact, it was slightly stabilized (Fig. 9B). These experiments indicate that KSHV ORF50 behaves with immediateearly kinetics on lytic cycle reactivation. Lytic cycle reactivation of KSHV, induced by Na butyrate, is likely to require modification of existing transcription factors and/or chromatin remodeling of the ORF50 promoter. Studies show that sodium butyrate treatment enhances the interaction of Sp1 protein with the ORF50 promoter (Lu et al., 2003; Ye et al., 2005). Fig. 9 Effect of CHX on EBV and KSHV lytic cycle induction by sodium butyrate (NaB) in dually infected BC 1 cells. BC 1 cells were untreated or treated with NaB. One set of cultures received CHX. RNA, harvested at intervals after addition of NaB, was analyzed for EBV BRLF1 or KSHV ORF50 by quantitative real time reverse transcriptase PCR. At 3, 5, and 8 h, EBV BRLF1 was inhibited by CHX. At 3 and 5 h, KSHV ORF50 was resistant to CHX. S.I., stimulation index.

17 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 97 In striking contrast, EBV BRLF1 (Rta) and BZLF1 (ZEBRA) require de novo protein synthesis before reactivation and are therefore not immediate early genes. One hypothesis is that EBV reactivation requires de novo protein synthesis of cellular transcription factor(s) that regulate the promoters of the BZLF1 and BRLF1 genes. C. Role of Phosphorylation in the Downstream Functions of the EBV ZEBRA Protein The preliminary unpublished experiments presented in Figs. 7 9 relate to upstream events leading to lytic cycle activation. As an example of downstream studies, we will describe how phosphorylation influences the function of the ZEBRA protein. ZEBRA is a member of the basic zipper (bzip) family of transcription factor proteins (Flemington and Speck, 1990; Kouzarides et al., 1991)(Fig. 10A). It shares homology with cellular bzip proteins such as c Jun, c Fos, and GCN4, especially in the basic DNA recognition domain. ZEBRA forms a homodimer in the absence of DNA. It binds specifically to AP 1 sites and to a large family of heterogenous ZRE (Lehman et al., 1998). ZEBRA can be considered to consist of five modular elements: a transcriptional activation domain (aa 1 167), a regulatory domain (aa ), a basic DNA recognition domain (aa ), a dimerization domain (aa ), and an accessory tail region that stabilizes the homodimer (aa ) (Petosa et al., 2006). ZEBRA is a phosphoprotein: it is constitutively phosphorylated in vivo at S167 and S173 [casein kinase 2 (CK2) sites], at T14 (a putative JNK site), and at S6, T7, S8 (El Guindy et al., 2006). It can be phosphorylated at T159 and S186 by PKC in vitro and in vivo when PKC is activated (Baumann et al., 1998). A report indicates that ZEBRA can also be phosphorylated by EBV protein kinase encoded in BGLF4 (Asai et al., 2006). ZEBRA performs many functions in the lytic viral life cycle (Fig. 10B): (1) it directly activates the BRLF1 (Rta) gene, (2) it synergizes with Rta in activating some downstream early genes, (3) it represses the activation of late genes by Rta at early times in the viral lytic cycle, and (4) it acts as an essential lytic viral replication protein (Fixman et al., 1992). Furthermore, ZEBRA impinges on many cellular pathways: (1) it activates MAP kinases such as p38 and JNK (Adamson et al., 2000), (2) it mediates cell cycle arrest (Cayrol and Flemington, 1996a,b), and (3) it exerts immunomodulatory effects by inhibiting the action of interferon, TNF and NF B (Morrison and Kenney, 2004; Morrison et al., 2001, 2004). It is of obvious interest to know which of these many functions of the ZEBRA protein are influenced by its phosphorylation state.

18 98 George Miller et al. Fig. 10 Functions of EBV ZEBRA protein. (A) Functional domains of the ZEBRA protein with identified phosphorylation sites. Constitutive phosphorylation by CK2 and JNK is shown in bold. Phosphorylation of ZEBRA by PKC is inducible (Baumann et al., 1998). Residues that are strongly phosphorylated in vitro (T14, S173, and S186) are shown in bold (El Guindy et al., 2006). (B) Four different functions of ZEBRA in EBV lytic cycle activation: (1) activation of transcription of the EBV BRLF1 promoter, (2) synergy with Rta in activation of the EBV BMRF1 promoter, (3) repression of Rta activation of the BLRF2 promoter at early times, and (4) as a lytic origin of DNA replication binding protein. Phosphorylation of ZEBRA at its CK2 sites, especially S173, influences its capacity to act as a repressor (El Guindy and Miller, 2004). A ZEBRA mutant Z(S173A) that cannot be phosphorylated by CK2 loses its capacity to repress the ability of Rta to activate a viral late gene BRLF2 at early times during the lytic cycle. An inhibitor of CK2, TBB, also blocks the capacity of ZEBRA to act as a repressor of Rta. Neither the mechanism of repression by ZEBRA, when phosphorylated at S173, nor its temporal control is well understood. At early times in the lytic cycle, when ZEBRA is phosphorylated at its major CK2 site, it may inhibit the activity of Rta or block the access of Rta to DNA. At late times in the lytic cycle, after DNA replicates,

19 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 99 ZEBRA no longer acts as a repressor of late gene activation by Rta. It is likely that the phosphorylation state of ZEBRA may be temporally regulated, with ZEBRA becoming dephosphorylated at its CK2 site after, or concomitant with, lytic viral DNA replication (Fig. 12A). Many other questions about the role of ZEBRA as a repressor remain to be investigated. Why is the repressive activity of ZEBRA, when phosphorylated by CK2, promoter specific? What differentiates promoters that are activated from those that are repressed by ZEBRA? Do ZEBRA proteins in different phosphorylated states act as activators and repressors? How does a constitutively expressed kinase, such as CK2, control the activity of a transcription factor? If the phosphorylation state of ZEBRA changes at different time points during the viral life cycle, as we postulate, is ZEBRA phosphorylation somehow related to lytic viral DNA replication? 1. CK2 SITE MUTANTS ARE SELECTIVELY DEFECTIVE AT ACTIVATING EBV LATE GENE EXPRESSION Ayman El Guindy recently investigated the role of the CK2 phosphorylation site in temporal control of EBV lytic gene expression by comparing viral early and late gene expression induced by wild type ZEBRA and a mutant ZEBRA, Z(S167A/S173A) in which both potential CK2 sites were changed to alanine. The experiments were conducted in BZKO cells which harbor an EBV genome containing an insertionally inactivated BZLF1 gene (Delecluse et al., 1999; Feederle et al., 2000). ZEBRA with mutant CK2 sites was equivalent to wild type ZEBRA protein in its ability to activate expression of viral early products, the transcription factor Rta, and the EA D (BMRF1) DNA polymerase processivity factor. However, the Z(S167A/ S173A) mutant was markedly defective at activating expression of a late small viral capsid protein encoded by EBV BFRF3 (Fig. 11A). We considered a number of possible explanations to account for this remarkable phenotype. (1) Since the CK2 site mutant fails to act as a repressor, it could permit or facilitate the accumulation of a product that inhibits late gene expression. (2) Since Z(S167A/S173A) activates the EBV early genes BRLF1 and BMRF1, a ZEBRA protein mutated at the CK2 site does not possess a general deficit in transcriptional activation. However, such a mutant might have a selective defect that renders it unable to activate specific viral early genes or cellular genes whose products are required for lytic viral DNA replication. (3) The CK2 site mutant ZEBRA protein might be unable to activate transcription of late genes. (4) Since transcription of late genes is dependent on lytic DNA replication, the CK2 site mutant might be deficient in promoting lytic viral DNA replication. El Guindy has obtained convincing evidence favoring the last hypothesis.

20 100 George Miller et al. Fig. 11 An alanine substitution mutant at ZEBRA s principal CK2 phosphorylation site is defective at inducing late gene expression and lytic viral DNA replication. (A) BZKO cells, which contain an insertionally inactivated BZLF1 gene, were transfected with CMV vector, ZEBRA, or Z(S167A/S173A) mutant. At 24 h intervals after transfection the cells were analyzed for latent, immediate early, early, or late EBV protein expression by immunoblotting with specific antibodies. (B) BZKO cells transfected with wild type or CK2 site mutant ZEBRA expression vectors were analyzed for EBV lytic DNA replication by Southern blotting with a probe adjacent to the viral terminal repeats (Raab Traub and Flynn, 1986).

21 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses CK2 SITE MUTANTS OF ZEBRA ARE IMPAIRED IN THEIR ABILITY TO ACTIVATE LYTIC VIRAL DNA REPLICATION Two complementary approaches were used to investigate the importance of the CK2 site of ZEBRA in activating lytic EB viral DNA replication. Using quantitative real time PCR, the total amount of EBV DNA in BZKO cells that had been transfected with an empty vector, or expression vectors for ZEBRA or the CK2 site mutant was measured. Wild type ZEBRA stimulated a 62 fold increase in viral DNA; the Z(S167A/S173A) mutant activated a sixfold increase. Southern blots were used to detect lytic DNA replication by probing for the ladder of DNA restriction fragments containing variable number of EBV terminal repeats (Fig. 11B). The ladder that reflects lytic replication was observed when BZKO cells were transfected with wild type ZEBRA or the Z(S167A) mutant. However, only a faint ladder was detected after transfection of the mutant Z(S173A) or the double mutant Z(S167A/S173A). These experiments showed that the CK2 site mutant was impaired in activation of lytic viral DNA replication and that residue S173 in the regulatory domain was principally involved in regulating lytic viral DNA synthesis. The question remains, what is the mechanism by which phosphorylation of ZEBRA at residue S173 licenses the protein to function in lytic DNA replication? ZEBRA is an origin binding protein. The EBV lytic origin of replication contains seven binding sites for ZEBRA, at least four of which are essential for the origin to function (Schepers et al., 1996). El Guindy has shown in three consecutive replicate chromatin immunoprecipitation experiments that the ZEBRA S173A mutant is approximately one third as active as wild type protein in binding to orilyt in vivo. In vitro electrophoretic mobility shift DNA binding experiments also demonstrate that the Z(S173A) mutant is markedly deficient by comparison to wild type at binding all seven of the ZRE in orilyt. Therefore, phosphorylation of ZEBRA at S173 achieves maximal EBV lytic DNA replication by promoting high DNA binding affinity. However, DNA binding affinity may not be the only property of ZEBRA that is regulated by phosphorylation at S173. In order to function as an essential EBV, lytic replication protein ZEBRA must possess at least two other functions. It must localize to the subcompartment of the nucleus, so called factories (Fig. 1B) where lytic viral DNA replication occurs. ZEBRA must also recruit and form a complex with other virally encoded replication proteins (Fig. 10B). Phosphorylation of ZEBRA at S173 may be required for the protein to fulfill these functions. The ZEBRA mutant S173A is fully competent to activate expression of Rta or diffuse early antigen (EA D) (Fig. 11A). Although it is possible that this mutant is specifically defective at activating expression of certain genes required for DNA replication, the current data support the view that

22 102 George Miller et al. phosphorylation at S173 is not essential for maximal transcriptional activation of viral early genes. El Guindy has done ChIP experiments to measure the association of wild type ZEBRA and the Z(S173A) mutant to Rp, the promoter of the BRLF1 (Rta) gene. Again the Z(S173A) mutant is about one third as active as wild type in associating with Rp in vivo. This interesting finding has two implications: first, phosphorylation of S173 has a general enhancing effect on DNA binding in vivo rather than a site specific effect. Second, since the CK2 site mutant of ZEBRA is impaired in replication but not in transcriptional activation, replication may be more sensitive than transcription to decreases in DNA binding affinity. The EBV origin of lytic replication contains seven contiguous ZEBRAbinding sites, while most ZEBRA responsive promoters contain 1, 2, or 3 sites. Thus, cooperative high affinity binding by ZEBRA may be needed to initiate DNA replication. The DNA binding requirement for recruitment of the transcription machinery may not be so stringent. 3. SUMMARY OF FUNCTIONAL ROLE OF CK2 PHOSPHORYLATION OF ZEBRA ON LYTIC CYCLE ACTIVATION Lytic cycle inducing stimuli that cause EBV to leave the latent state lead to expression of the viral ZEBRA and Rta proteins. These two transcription factors synergize to activate most early lytic genes. Early genes encode Fig. 12 Models for the role of phosphorylation of ZEBRA at its CK2 site in the temporal control of the EBV lytic cycle. (A) At early times in the lytic cycle, when ZEBRA is phosphorylated at S173, it acts as a repressor of Rta s capacity to activate an EBV late gene. At late times, ZEBRA may become dephosphorylated and no longer acts as a repressor. (B) At early times in the lytic cycle when ZEBRA is phosphorylated at S173, it can function optimally as an origin binding protein and promote lytic viral DNA replication.

23 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 103 products required for DNA replication. Lytic replication is needed for late gene expression. Rta has the capacity to activate expression of some late genes at early times in the viral lytic cycle. Such aberrant late gene expression may inhibit DNA replication by a feedback pathway. Phosphorylation of ZEBRA at its CK2 site in the regulatory domain maximizes the DNA binding affinity of ZEBRA. ZEBRA in its phosphorylated high affinity DNA binding state performs two functions that are critical for proper temporal control of the EBV life cycle. Phosphorylated ZEBRA represses the activation of Rta of late genes at early times. Phosphorylated ZEBRA acts as a lytic origin binding protein, thus facilitating lytic viral DNA replication (Fig. 12B). III. CONCLUSIONS: SOME UNSOLVED MYSTERIES ABOUT LYTIC CYCLE SWITCHES OF ONCOGENIC HUMAN GAMMAHERPESVIRUSES In this essay, we classify the events leading to lytic cycle activation either as upstream events, resulting in repression or activation of the promoters of the lytic cycle activator genes of EBV and KSHV, and downstream events, resulting from activation or repression of viral lytic gene promoters and viral lytic origins of replication by the lytic cycle activator proteins, the ZEBRA and Rta proteins of EBV and ORF50 protein of KSHV. We have pointed out many, yet to be resolved, mysteries about this deceptively simple biologic system. Among the mysteries about upstream events, we would mention the following: why do EBV and KSHV genomes exist as multicopy plasmids? When lytic induction occurs, does each genome respond or are the genomes heterogeneous in their response? Although the lytic cycle can be manipulated in cultured B cells, we know very little about the physiologic stimuli that repress or activate the lytic cycle in vivo. Is the physiologic signal external, mediated by cytokines, hormones, or cells of the immune system, or does lytic cycle activation occur as a cellular response to stress such as hypoxia or change in energy charge of the cell? Not all cultured cell lines respond to the same inducing stimulus. Some cell lines respond to inducing stimuli, such as PKC agonists, HDAC inhibitors, and DNA methyltransferase inhibitors, which act via quite distinct mechanisms. These observations raise several questions about lytic cycle activation in cultured cells. What accounts for differences in response of different cell lines to inducing stimuli? Do lytic cycle inducing agents that act by different biochemical mechanisms operate via a common pathway or are there several distinct routes to lytic cycle activation?

24 104 George Miller et al. There are many unresolved issues about the mechanism of action of inducing stimuli. For example, is the action of HDAC inhibitors, such as sodium butyrate or Trichostatin A, mediated solely by opening chromatin on the promoters of the lytic cycle activator genes or are additional events required? In preliminary data, we show that the DNA methyltransferase inhibitor 5Aza2 0 dc rapidly induces lytic cycle gene expression without detectably altering the methylation state or acetylation of histone tails on the promoters of the lytic cycle activator genes. What additional property of 5Aza2 0 dc is responsible for this rapid effect? Even in the most responsive cell line exposed to the most potent inducing stimulus only a subpopulation of the cells respond. What accounts for the refractory subpopulation? What mediates the refractory state? Are some cells permanently marked for lack of response or is the refractory state transient? In one set of experiments (Figs. 8 and 9), we ask whether inducing stimuli activate transcription factors that are already present in the cell, or whether these transcription factors need to be synthesized before lytic cycle activation can occur. We find, surprisingly, that CHX, an inhibitor of protein synthesis, blocks lytic cycle induction of EBV but not KSHV. Thus this question has two different answers for the two gammaherpesviruses. This result poses another important question: What protein(s) need to be synthesized in order for EBV ZEBRA and Rta to be made? A central unanswered question about the downstream events is why EBV employs two different transcription factors, ZEBRA and Rta, to control expression of lytic cycle genes, whereas KSHV employs only one, ORF50 the homologue of Rta? What is the mechanism of synergy between ZEBRA and Rta? The EBV ZEBRA protein plays two distinct roles: as a transcription factor and as a DNA replication protein. The ZEBRA homologue in KSHV, that is K8 bzip, is mainly a DNA replication protein. What features of the two bzip proteins account for these differences in biologic activity? ZEBRA acts both as an activator and as a repressor of the promoters of lytic cycle genes. The capacity of ZEBRA to act as a repressor of late genes is mediated by phosphorylation of residue S173. How does phosphorylation mediate this activity? How is phosphorylation temporally regulated? What mechanism accounts for the promoter specificity of repression? Finally, how does phosphorylation of ZEBRA license its role as an origin binding protein? Does enhanced DNA binding activity suffice to explain the role of phosphorylated ZEBRA in DNA replication? Does phosphorylation of ZEBRA affect interactions with cellular and viral replication proteins? Does phosphorylation of ZEBRA alter its subnuclear localization? The search for answers to these and many other questions makes study of the lytic cycle switches of the oncogenic human herpesviruses a fascinating and fertile field.

25 Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses 105 ACKNOWLEDGMENTS Work in G.M. s laboratory was supported by grants from the United States National Cancer Institute: CA12055, CA16038, CA70036, and CA This essay emphasizes work in the authors laboratories and is not intended as a formal review of the published literature. We thank Susan Prisley and Karen Lavery for assistance in the preparation of the figures and chapter. We thank H. J. Delecluse for the BZKO cells. REFERENCES Adamson, A. L., Darr, D., Holley Guthrie, E., Johnson, R. A., Mauser, A., Swenson, J., and Kenney, S. (2000). Epstein Barr virus immediate early proteins BZLF1 and BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphorylated p38 and c Jun N terminal kinases. J. Virol. 74(3), Armstrong, D., Henle, G., and Henle, W. (1966). Complement fixation tests with cell lines derived from Burkitt s lymphoma and acute leukemias. J. Bacteriol. 91(3), Asai, R., Kato, A., Kato, K., Kanamori Koyama, M., Sugimoto, K., Sairenji, T., Nishiyama, Y., and Kawaguchi, Y. (2006). Epstein Barr virus protein kinase BGLF4 is a virion tegument protein that dissociates from virions in a phosphorylation dependent process and phosphorylates the viral immediate early protein BZLF1. J. Virol. 80(11), Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H. J., and Hammerschmidt, W. (1998). Activation of the Epstein Barr virus transcription factor BZLF1 by 12 O tetradecanoylphorbol 13 acetate induced phosphorylation. J. Virol. 72(10), Baumann, M., Feederle, R., Kremmer, E., and Hammerschmidt, W. (1999). Cellular transcription factors recruit viral replication proteins to activate the Epstein Barr virus origin of lytic DNA replication, orilyt. [published erratum appears in EMBO J Jan 17; 19(2), 315]. EMBO J. 18(21), Ben Sasson, S. A., and Klein, G. (1981). Activation of the Epstein Barr virus genome by 5 azacytidine in latently infected human lymphoid lines. Int. J. Cancer 28(2), Bhende, P. M., Seaman, W. T., Delecluse, H. J., and Kenney, S. C. (2004). The EBV lytic switch protein, Z, preferentially binds to and activates the methylated viral genome. Nat. Genet. 36(10), Bhende, P. M., Seaman, W. T., Delecluse, H. J., and Kenney, S. C. (2005). BZLF1 activation of the methylated form of the BRLF1 immediate early promoter is regulated by BZLF1 residue 186. J. Virol. 79(12), Cayrol, C., and Flemington, E. (1996a). G0/G1 growth arrest mediated by a region encompassing the basic leucine zipper (bzip) domain of the Epstein Barr virus transactivator Zta. J. Biol. Chem. 271(50), Cayrol, C., and Flemington, E. K. (1996b). The Epstein Barr virus bzip transcription factor Zta causes G0/G1 cell cycle arrest through induction of cyclin dependent kinase inhibitors. EMBO J. 15(11), Cesarman, E., Chang, Y., Moore, P. S., Said, J. W., and Knowles, D. M. (1995). Kaposi s sarcoma associated herpesvirus like DNA sequences in AIDS related body cavity based lymphomas. N. Engl. J. Med. 332(18), Chang, P. J., Shedd, D., Gradoville, L., Cho, M. S., Chen, L. W., Chang, J., and Miller, G. (2002). Open reading frame 50 protein of Kaposi s sarcoma associated herpesvirus directly