Identifying the Cellular Interactome of Epstein-Barr Virus Lytic Regulator Zta Reveals Cellular Targets Contributing to Viral Replication.

The human gammaherpesvirus Epstein-Barr virus (EBV) (human herpesvirus 4 [HHV4]) infects most adults and is an important contributor to the development of many types of lymphoid and epithelial cancers. Essential contributions of viral genes to viral replication are known, but the potential contributions of cell genes are less well delineated. A key player is the viral protein Zta (BZLF1, ZEBRA, or Z). This sequence-specific DNA-binding protein can disrupt EBV latency by driving the transcription of target genes and by interacting with the EBV lytic origin of replication. Here, we used an unbiased proteomics approach to identify the Zta-interactome in cells derived from Burkitt's lymphoma. Isolating Zta and associated proteins from Burkitt's lymphoma cells undergoing EBV replication, followed by tandem mass tag (TMT) mass spectrometry, resulted in the identification of 39 viral and cellular proteins within the Zta interactome. An association of Zta with the cellular protein NFATc2 was validated in independent experiments. Furthermore, the ability of Zta to attenuate the activity of an NFAT-dependent promoter was shown, which suggests a functional consequence for the association. The expression of Zta is itself regulated through NFAT activity, suggesting that Zta may contribute to a feedback loop that would limit its own expression, thus aiding viral replication by preventing the known toxic effects of Zta overexpression.IMPORTANCE Epstein-Barr virus infects most people across the world and causes several kinds of cancer. Zta is an important viral protein that makes the virus replicate by binding to its DNA and turning on the expression of some genes. We used a sensitive, unbiased approach to isolate and identify viral and cellular proteins that physically interact with Zta. This revealed 39 viral and cellular proteins. We found that one protein, termed NFATc2, was already known to be important for a very early step in viral replication. We identify that once this step has occurred, Zta reduces the effectiveness of NFATc2, and we suggest that this is important to prevent cells from dying before viral replication is complete and the mature virus is released from the cells.

Mechanism of activation of the BNLF2a immune evasion gene of Epstein-Barr virus by Zta.

The human gamma herpes virus Epstein-Barr virus (EBV) exploits multiple routes to evade the cellular immune response. During the EBV lytic replication cycle, viral proteins are expressed that provide excellent targets for recognition by cytotoxic T cells. This is countered by the viral BNLF2a gene. In B cells during latency, where BNLF2a is not expressed, we show that its regulatory region is embedded in repressive chromatin. The expression of BNLF2a mirrors the expression of a viral lytic cycle transcriptional regulator, Zta (BZLF1, EB1, ZEBRA), in B cells and we propose that Zta plays a role in up-regulating BNLF2a. In cells undergoing EBV lytic replication, we identified two distinct regions of interaction of Zta with the chromatin-associated BNLF2a promoter. We identify five potential Zta-response elements (ZREs) in the promoter that are highly conserved between virus isolates. Zta binds to these elements in vitro and activates the expression of the BNLF2a promoter in both epithelial and B cells. We also found redundancy amongst the ZREs. The EBV genome undergoes a biphasic DNA methylation cycle during its infection cycle. One of the ZREs contains an integral CpG motif. We show that this can be DNA methylated during EBV latency and that both Zta binding and promoter activation are enhanced by its methylation. In summary, we find that the BNLF2a promoter is directly targeted by Zta and that DNA methylation within the proximal ZRE aids activation. The implications for regulation of this key viral gene during the reactivation of EBV from latency are discussed.

Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements.

Repression of CIITA by the Epstein-Barr virus transcription factor Zta is independent of its dimerization and DNA binding.

Repression of the cellular CIITA gene is part of the immune evasion strategy of the γherpes virus Epstein-Barr virus (EBV) during its lytic replication cycle in B-cells. In part, this is mediated through downregulation of MHC class II gene expression via the targeted repression of CIITA, the cellular master regulator of MHC class II gene expression. This repression is achieved through a reduction in CIITA promoter activity, initiated by the EBV transcription and replication factor, Zta (BZLF1, EB1, ZEBRA). Zta is the earliest gene expressed during the lytic replication cycle. Zta interacts with sequence-specific elements in promoters, enhancers and the replication origin (ZREs), and also modulates gene expression through interaction with cellular transcription factors and co-activators. Here, we explore the requirements for Zta-mediated repression of the CIITA promoter. We find that repression by Zta is specific for the CIITA promoter and can be achieved in the absence of other EBV genes. Surprisingly, we find that the dimerization region of Zta is not required to mediate repression. This contrasts with an obligate requirement of this region to correctly orientate the DNA contact regions of Zta to mediate activation of gene expression through ZREs. Additional support for the model that Zta represses the CIITA promoter without direct DNA binding comes from promoter mapping that shows that repression does not require the presence of a ZRE in the CIITA promoter.

Induction of p16(INK4a) is the major barrier to proliferation when Epstein-Barr virus (EBV) transforms primary B cells into lymphoblastoid cell lines.

To explore the role of p16(INK4a) as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16(INK4a) and p14(ARF). Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16(INK4a) protein but expressing a functional 14(ARF)-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C--in cooperation with EBNA3A--despite the absence of functional p16(INK4a). Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16(INK4a) and growth arrest, EBNA3C inactivation in the p16(INK4a)-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16(INK4a)-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16(INK4a) expression and concomitant block to proliferation 2-4 weeks post-infection. If cells are p16(INK4a)-null, functional EBNA3C is dispensable for the outgrowth of LCLs.

Dynamic chromatin environment of key lytic cycle regulatory regions of the Epstein-Barr virus genome.

The ability of Epstein-Barr virus (EBV) to establish latency allows it to evade the immune system and to persist for the lifetime of its host; one distinguishing characteristic is the lack of transcription of the majority of viral genes. Entry into the lytic cycle is coordinated by the viral transcription factor, Zta (BZLF1, ZEBRA, and EB1), and downstream effectors, while viral genome replication requires the concerted action of Zta and six other viral proteins at the origins of lytic replication. We explored the chromatin context at key EBV lytic cycle promoters (BZLF1, BRLF1, BMRF1, and BALF5) and the origins of lytic replication during latency and lytic replication. We show that a repressive heterochromatin-like environment (trimethylation of histone H3 at lysine 9 [H3K9me3] and lysine 27 [H3K27me3]), which blocks the interaction of some transcription factors with DNA, encompasses the key early lytic regulatory regions. Epigenetic silencing of the EBV genome is also imposed by DNA methylation during latency. The chromatin environment changes during the lytic cycle with activation of histones H3, H4, and H2AX occurring at both the origins of replication and at the key lytic regulatory elements. We propose that Zta is able to reverse the effects of latency-associated repressive chromatin at EBV early lytic promoters by interacting with Zta response elements within the H3K9me3-associated chromatin and demonstrate that these interactions occur in vivo. Since the interaction of Zta with DNA is not inhibited by DNA methylation, it is clear that Zta uses two routes to overcome epigenetic silencing of its genome.

Genome-wide analyses of Zta binding to the Epstein-Barr virus genome reveals interactions in both early and late lytic cycles and an epigenetic switch leading to an altered binding profile.

The Epstein-Barr virus (EBV) genome sustains substantial epigenetic modification involving chromatin remodelling and DNA methylation during lytic replication. Zta (ZEBRA, BZLF1), a key regulator of the EBV lytic cycle, is a transcription and replication factor, binding to Zta response elements (ZREs) in target promoters and EBV lytic origins of replication. In vitro, Zta binding is modulated by DNA methylation; a subset of CpG-containing Zta binding sites (CpG ZREs) is bound only in a DNA methylation-dependent manner. The question of how the dynamic epigenetic environment impacts Zta interaction during the EBV lytic cycle is unknown. To address this, we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) to identify Zta binding sites across the EBV genome before and after viral DNA replication. Replication did not alter the association of Zta across many regions of the EBV genome, but a striking reduction in Zta binding occurred at some loci that contain CpG ZREs. Separating Zta-bound DNA into methylated and nonmethylated fractions, we found that promoters that contain CpG ZREs were enriched in the methylated fraction but that Zta binding to promoters lacking CpG ZREs was not reduced. We hypothesize that the loss of DNA methylation on the EBV genome during the lytic cycle causes the reduced binding to CpG ZREs; this may act as a lytic cycle epigenetic switch. However, the epigenetic changes associated with the replicated EBV genome do not affect the interaction of Zta with many loci that are rich in non-CpG ZREs; this leads to sustained binding at these regions.

Could Changing the DNA Methylation Landscape Promote the Destruction of Epstein-Barr Virus-Associated Cancers?

DNA methylation at CpG motifs provides an epigenetic route to regulate gene expression. In general, an inverse correlation between DNA hypermethylation at CpG motifs and gene expression is observed. Epstein Barr-virus (EBV) infects people and the EBV genome resides in the nucleus where either its replication cycle initiates or it enters a long-term latency state where the viral genome becomes hypermethylated at CpG motifs. Viral gene expression shows a largely inverse correlation with DNA hypermethylation. DNA methylation occurs through the action of DNA methyl transferase enzymes: writer DNA methyl transferases add methyl groups to specific regions of unmethylated DNA; maintenance DNA methyl transferases reproduce the pattern of DNA methylation during genome replication. The impact of DNA methylation is achieved through the association of various proteins specifically with methylated DNA and their influence on gene regulation. DNA methylation can be changed through altering DNA methyl transferase activity or through the action of enzymes that further modify methylated CpG motifs. Azacytidine prodrugs that are incorporated into CpG motifs during DNA replication are recognized by DNA methyl transferases and block their function resulting in hypomethylation of DNA. EBV-associated cancers have hypermethylated viral genomes and many carcinomas also have highly hypermethylated cellular genomes. Decitabine, a member of the azacytidine prodrug family, reactivates viral gene expression and promotes the recognition of lymphoma cells by virus-specific cytotoxic T-cells. For EBV-associated cancers, the impact of decitabine on the cellular genome and the prospect of combining decitabine with other therapeutic approaches is currently unknown but exciting.

Interaction sites of the Epstein-Barr virus Zta transcription factor with the host genome in epithelial cells.

Epstein-Barr virus (EBV) is present in a state of latency in infected memory B-cells and EBV-associated lymphoid and epithelial cancers. Cell stimulation or differentiation of infected B-cells and epithelial cells induces reactivation to the lytic replication cycle. In each cell type, the EBV transcription and replication factor Zta (BZLF1, EB1) plays a role in mediating the lytic cycle of EBV. Zta is a transcription factor that interacts directly with Zta response elements (ZREs) within viral and cellular genomes. Here we undertake chromatin-precipitation coupled to DNA-sequencing (ChIP-Seq) of Zta-associated DNA from cancer-derived epithelial cells. The analysis identified over 14 000 Zta-binding sites in the cellular genome. We assessed the impact of lytic cycle reactivation on changes in gene expression for a panel of Zta-associated cellular genes. Finally, we compared the Zta-binding sites identified in this study with those previously identified in B-cells and reveal substantial conservation in genes associated with Zta-binding sites.

Is EBV Associated with Breast Cancer in Specific Geographic Locations?

Epstein-Barr virus (EBV) is a virus that establishes a life-long infection in people, and infection with EBV is nearly ubiquitous by adulthood. EBV was identified from biopsy material from a child with Burkitt's lymphoma (BL) in sub-Saharan Africa. EBV has a well-characterised role in the development of some cancers, notably, Burkitt's lymphoma (BL), Hodgkin's disease (HD), gastric carcinoma (GC), and nasopharyngeal carcinoma (NPC). Links have also been made between EBV and breast cancer (BC), but these have been controversial. For all EBV-associated cancers, the ubiquitous nature of infection with EBV, contrasted with the relatively rare development of cancer, highlights a problem of determining whether EBV is an aetiological agent of cancer. In addition, the geographic distributions of some EBV-associated cancers point to contributions from additional co-factors. Recent meta-analyses of the incidence of EBV within BC biopsies has revealed that the diversity in the conclusions remain, however, they also show more of an association between EBV and BC biopsies in some study locations. Here, we review the evidence linking EBV with BC, and conclude that the evidence for the presence of EBV in BC biopsies is concentrated in specific geographic regions but is currently insufficient to provide a causal link. We pose some questions that could help to resolve the question of whether EBV contributes to BC and probe the contribution EBV might make to the aetiology of BC.

Functional interaction between Epstein-Barr virus replication protein Zta and host DNA damage response protein 53BP1.

Epstein-Barr virus (EBV; human herpesvirus 4) poses major clinical problems worldwide. Following primary infection, EBV enters a form of long-lived latency in B lymphocytes, expressing few viral genes, and it persists for the lifetime of the host with sporadic bursts of viral replication. The switch between latency and replication is governed by the action of a multifunctional viral protein Zta (also called BZLF1, ZEBRA, and Z). Using a global proteomic approach, we identified a host DNA damage repair protein that specifically interacts with Zta: 53BP1. 53BP1 is intimately connected with the ATM signal transduction pathway, which is activated during EBV replication. The interaction of 53BP1 with Zta requires the C-terminal ends of both proteins. A series of Zta mutants that show a wild-type ability to perform basic functions of Zta, such as dimer formation, interaction with DNA, and the transactivation of viral genes, were shown to have lost the ability to induce the viral lytic cycle. Each of these mutants also is compromised in the C-terminal region for interaction with 53BP1. In addition, the knockdown of 53BP1 expression reduced viral replication, suggesting that the association between Zta and 53BP1 is involved in the viral replication cycle.

Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

Unexpected structure of Epstein-Barr virus lytic cycle activator Zta.

The key viral gene responsible for initiating the replicative cycle of Epstein-Barr virus (EBV), termed BZLF1, encodes the multifunctional protein Zta (ZEBRA or Z). It interacts with DNA as both a transcription and a replication factor, modulates both intracellular signal transduction and the DNA-damage response and manipulates cell cycle progression. Muller and colleagues have resolved the structure of Zta bound to DNA, which confirms some structural predictions but reveals an unexpected twist and a complex dimerization interface. Because EBV is associated with human disease, Zta presents a prime target for drug design.

The Use of Chromatin Precipitation Coupled to DNA Sequencing (ChIP-Seq) for the Analysis of Zta Binding to the Human and EBV Genome.

Determining which components of the transcription machinery associate with the viral and cellular genome, and how this changes at specific stages of the viral life cycle is paramount to understanding how the distinct transcriptional programs associated with primary infection, latency, and disease are established and how they are reprogrammed during initiation and execution of the viral lytic replication cycle. Chromatin precipitations linked to next generation DNA sequencing (ChIP-Seq) allow for the interactions of proteins with DNA to be mapped across both viral and cellular genomes. This can be applied to viral and cellular transcription factors, coactivators and corepressors, modified histones, and modulators of chromatin.

Identification of Epstein-Barr Virus Replication Proteins in Burkitt's Lymphoma Cells.

The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications.

Epigenetic control of Epstein-Barr virus transcription - relevance to viral life cycle?

DNA methylation normally leads to silencing of gene expression but Epstein-Barr virus (EBV) provides an exception to the epigenetic paradigm. DNA methylation is absolutely required for the expression of many viral genes. Although the viral genome is initially un-methylated in newly infected cells, it becomes extensively methylated during the establishment of viral latency. One of the major regulators of EBV gene expression is a viral transcription factor called Zta (BZLF1, ZEBRA, Z) that resembles the cellular AP1 transcription factor. Zta recognizes at least 32 variants of a 7-nucleotide DNA sequence element, the Zta-response element (ZRE), some of which contain a CpG motif. Zta only binds to the latter class of ZREs in their DNA-methylated form, whether they occur in viral or cellular promoters and is functionally relevant for the activity of these promoters. The ability of Zta to interpret the differential DNA methylation of the viral genome is paramount for both the establishment of viral latency and the release from latency to initiate viral replication.

Epigenetic control of viral life-cycle by a DNA-methylation dependent transcription factor.

Epstein-Barr virus (EBV) encoded transcription factor Zta (BZLF1, ZEBRA, EB1) is the prototype of a class of transcription factor (including C/EBPalpha) that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs) has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.

Evaluation of a prediction protocol to identify potential targets of epigenetic reprogramming by the cancer associated Epstein Barr virus.

BACKGROUND: Epstein Barr virus (EBV) infects the majority of the human population, causing fatal diseases in a small proportion in conjunction with environmental factors. Following primary infection, EBV remains latent in the memory B cell population for life. Recurrent reactivation of the virus occurs, probably due to activation of the memory B-lymphocytes, resulting in viral replication and re-infection of B-lymphocytes. Methylation of the viral DNA at CpG motifs leads to silencing of viral gene expression during latency. Zta, the key viral protein that mediates the latency/reactivation balance, interacts with methylated DNA. Zta is a transcription factor for both viral and host genes. A sub-set of its DNA binding sites (ZREs) contains a CpG motif, which is recognised in its methylated form. Detailed analysis of the promoter of the viral gene BRLF1 revealed that interaction with a methylated CpG ZRE (RpZRE3) is key to overturning the epigenetic silencing of the gene. METHODOLOGY AND PRINCIPAL FINDINGS: Here we question whether we can use this information to identify which host genes contain promoters with similar response elements. A computational search of human gene promoters identified 274 targets containing the 7-nucleotide RpZRE3 core element. DNA binding analysis of Zta with 17 of these targets revealed that the flanking context of the core element does not have a profound effect on the ability of Zta to interact with the methylated sites. A second juxtaposed ZRE was observed for one promoter. Zta was able to interact with this site, although co-occupancy with the RpZRE3 core element was not observed. CONCLUSIONS/SIGNIFICANCE: This research demonstrates 274 human promoters have the potential to be regulated by Zta to overturn epigenetic silencing of gene expression during viral reactivation from latency.

The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1.

Activation of the host gene egr1 is essential for the lytic replication of Epstein-Barr virus (EBV). egr1 is activated by Zta (BZLF1, ZEBRA). Zta interacts directly with DNA through a series of closely related Zta-response elements (ZREs). Here we dissect the mechanism used by Zta to interact with the egr1 promoter and identify a weak interaction with egr1ZRE that is dependent on the distal part of egr1ZRE. Furthermore, we demonstrate that the ability of Zta to interact with egr1ZRE is enhanced at least tenfold by methylation. The ability of Zta to transactivate a reporter construct driven by the egr1 promoter can be enhanced by methylation. As the ability of Zta to interact with a methylated ZRE in the EBV genome correlates with its ability to activate the expression of the endogenous viral gene BRLF1, this suggests that Zta may also have the capability to overturn epigenetic control of egr1.

The reversal of epigenetic silencing of the EBV genome is regulated by viral bZIP protein.

EBV (Epstein-Barr virus) alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and in the switch to the lytic cycle this epigenetic silencing is overturned. A key event is the activation of the viral protein Zta with three ZREs (Zta-response elements) from the BRLF1 promoter (referred to as Rp). Two of these ZREs contain CpG motifs and are methylated in the latent genome. Biochemical analyses and molecular modelling of Zta bound to methylated RpZRE3 indicate the precise contacts made between a serine and a cysteine residue of Zta with methyl cytosines. A single point mutant of Zta, C189S, is defective in binding to the methylated ZREs both in vitro and in vivo. This was used to probe the functional relevance of the interaction. ZtaC189S was not able to activate Rp in a B-cell line, demonstrating the relevance of the interaction with methylated ZREs. This demonstrates that Zta plays a role in overturning the epigenetic control of viral latency.