Regulation of Epstein-Barr Virus Lytic Cycle Activation in Malignant and Nonmalignant Disease

cThe 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.

Section: Pstein-barr Virus (Ebv) Is a Ubiquitous Human Herpesvirusmentioning

confidence: 99%

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

Ramasubramanyan

Kanhere

Osborn

et al. 2012

“…During EBV replication, most viral genes are expressed, the EBV genome is replicated by viral enzymes, and infectious virions are produced. This cascade of EBV lytic gene expression is controlled by two viral transcription factors, Zta (Z, ZEBRA) and Rta (R), encoded by the BZLF1 and BRLF1 genes, respectively (50,61). Deletion of either BZLF1 or BRLF1 renders the virus incompetent for DNA replication and virion production (21).…”

mentioning

confidence: 99%

“…Late transcription follows DNA replication and can be blocked by inhibitors of DNA replication, such as phosphonoacetic acid (PAA) (37,38). Lytic gene promoters vary in their responsiveness to Zta and Rta (61). Some genes are activated primarily by Rta and others primarily by Zta, and some are synergistically activated by Zta and Rta (15,21,25,26,41,46,57,61,65).…”

mentioning

confidence: 99%

Genome-Wide Analysis of Epstein-Barr Virus Rta DNA Binding

Heilmann

Calderwood

Portal

et al. 2012

The Epstein-Barr virus (EBV) lytic transactivator Rta activates promoters through direct binding to cognate DNA sites termed Rta response elements (RREs). Rta also activates promoters that apparently lack Rta binding sites, notably Zp and Rp. Chromatin immunoprecipitation (ChIP) of endogenous Rta expressed during early replication in B95-8 cells was performed to identify Rta binding sites in the EBV genome. Quantitative PCR (qPCR) analysis showed strong enrichment for known RREs but little or no enrichment for Rp or Zp, suggesting that the Rta ChIP approach enriches for direct Rta binding sites. Rta ChIP combined with deep sequencing (ChIP-seq) identified most known RREs and several novel Rta binding sites. Rta ChIP-seq peaks were frequently upstream of Rta-responsive genes, indicating that these Rta binding sites are likely functioning as RREs. Unexpectedly, the BALF5 promoter contained an Rta binding peak. To assess whether BALF5 might be activated by an RRE-dependent mechanism, an Rta mutant (Rta K156A), deficient for DNA binding and RRE activation but competent for Zp/Rp activation, was used. Rta K156A failed to activate BALF5p, suggesting this promoter can be activated by an RRE-dependent mechanism. Rta binding to late gene promoters was not seen at early time points but was specifically detected at later times within the Rta-responsive BLRF2 and BFRF3 promoters, even when DNA replication was inhibited. Our results represent the first characterization of Rta binding to the EBV genome during replication, identify previously unknown RREs, such as one in BALF5p, and highlight the complexity of EBV late gene promoter activation by Rta.

“…Entry into replication is regulated by the EBV genes BZLF1 and BRLF1, encoding the transcriptional activators Z (Zta) and R (Rta), respectively (44,53). Zta and Rta must act in concert for EBV replication to occur: deletion of either BZLF1 or BRLF1 renders the virus incompetent for DNA replication and virion production (20).…”

mentioning

confidence: 99%

“…Zta and Rta must act in concert for EBV replication to occur: deletion of either BZLF1 or BRLF1 renders the virus incompetent for DNA replication and virion production (20). Some EBV lytic genes are activated primarily by Rta, others primarily by Zta, and some are synergistically activated by the combined actions of Rta and Zta (13,20,24,27,37,43,48,49,53,60,64). A subset of EBV genes is activated by Rta but repressed by Zta (18,49,64).…”

mentioning

confidence: 99%

Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization

Heilmann

Calderwood

Johannsen

2010

The switch from Epstein-Barr virus (EBV) latent infection to lytic replication is governed by two viral transactivators, Zta and Rta. We previously reported that the EBV protein LF2 binds Rta, inhibits Rta promoter activation, and blocks EBV replication in cells. In addition, LF2 induces SUMO2/3 modification of Rta. We now show that this modification occurs at four lysines within the Rta activation domain (426, 446, 517, and 530) and that sumoylation of Rta is not essential for its repression. Coexpression studies demonstrated that Rta is sequestered to the extranuclear cytoskeleton in the presence of LF2. We mapped the LF2 binding site to Rta amino acids (aa) 476 to 519 and showed that LF2 binding is critical for Rta relocalization and repression. The core of this binding site, Rta aa 500 to 526, confers LF2-mediated relocalization and repression onto the artificial transcription factor GAL4-VP16. Mutational analysis of LF2 provided further evidence that Rta redistribution is essential for repression. Rta localization changes during replication of the LF2-positive P3HR1 genome, but not during replication of the LF2-negative B95-8 genome. BLRF2 protein expression was decreased and delayed in P3HR1 cells compared with B95-8 cells, consistent with reduced Rta activity. By contrast, BMRF1 expression, regulated primarily by Zta, did not differ significantly between the two cell lines. Our results support a model in which LF2 regulates EBV replication by binding to Rta and redistributing it out of the nucleus. Epstein-Barr virus (EBV), the prototypical gammaherpesvirus, causes infectious mononucleosis in healthy individuals, B-cell lymphoproliferative disease in immunosuppressed individuals, and rarely, B-cell lymphomas, Hodgkin lymphoma, and nasopharyngeal carcinoma in otherwise-healthy persons (45, 65). Gammaherpesviruses, including EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), differ from other viruses because their associated diseases are not the consequence of virus replication. Instead, EBV-associated malignancies are a by-product of the growth and survival signals triggered by limited viral gene expression that allows EBV to persist in a latent state in infected cells, and hence the human population (44). Because viral replication is not occurring in most EBV-infected cells, inhibitors of replication are not efficacious in treating infectious mononucleosis or EBV-associated malignancies. On the contrary, activation of EBV replication has been suggested as therapy, because virus replication can directly kill EBV-infected tumor cells, sensitize them to nucleoside analogues, and stimulate immunemediated killing via increased virus antigen expression in tumor cells (21,22,55).Entry into replication is regulated by the EBV genes BZLF1 and BRLF1, encoding the transcriptional activators Z (Zta) and R (Rta), respectively (44, 53). Zta and Rta must act in concert for EBV replication to occur: deletion of either BZLF1 or BRLF1 renders the virus incompetent for DNA replication and virion production (20). Some E...