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Genome-wide analysis of Epstein-Barr virus Rta DNA binding.

ABSTRACT: 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.

SUBMITTER: Heilmann AM

PROVIDER: S-EPMC3347379 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Genome-wide analysis of Epstein-Barr virus Rta DNA binding.

Heilmann Andreas M F AM Calderwood Michael A MA Portal Daniel D Lu Yong Y Johannsen Eric E

Journal of virology 20120229 9

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 ...[more]

PMID: 22379087

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Project description:Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.

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Project description:The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children.

Dataset Information

Genome-wide analysis of Epstein-Barr virus Rta DNA binding.

Publications

Genome-wide analysis of Epstein-Barr virus Rta DNA binding.

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