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The B-cell-specific transcription factor and master regulator Pax5 promotes Epstein-Barr virus latency by negatively regulating the viral immediate early protein BZLF1.

ABSTRACT: The latent-to-lytic switch of Epstein-Barr virus (EBV) is mediated by the immediate early protein BZLF1 (Z). However, the cellular factors regulating this process remain incompletely characterized. In this report, we show that the B-cell-specific transcription factor Pax5 helps to promote viral latency in B cells by blocking Z function. Although Z was previously shown to directly interact with Pax5 and inhibit its activity, the effect of Pax5 on Z function has not been investigated. Here, we demonstrate that Pax5 inhibits Z-mediated lytic viral gene expression and the release of infectious viral particles in latently infected epithelial cell lines. Conversely, we found that shRNA-mediated knockdown of endogenous Pax5 in a Burkitt lymphoma B-cell line leads to viral reactivation. Furthermore, we show that Pax5 reduces Z activation of early lytic viral promoters in reporter gene assays and inhibits Z binding to lytic viral promoters in vivo. We confirm that Pax5 and Z directly interact and show that this interaction requires the carboxy-terminal DNA-binding/dimerization domain of Z and the amino-terminal DNA-binding domain of Pax5. A Pax5 DNA-binding mutant (V26G/P80R) that interacts with Z retains the ability to inhibit Z function, whereas a Pax5 mutant (Δ106-110) that is deficient for interaction with Z does not inhibit Z-mediated lytic viral reactivation. Since the B-cell-specific transcription factor Oct-2 also directly interacts with Z and inhibits its function, these results suggest that EBV uses multiple redundant mechanisms to establish and maintain viral latency in B cells.

SUBMITTER: Raver RM

PROVIDER: S-EPMC3700198 | biostudies-other | 2013 Jul

REPOSITORIES: biostudies-other

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The B-cell-specific transcription factor and master regulator Pax5 promotes Epstein-Barr virus latency by negatively regulating the viral immediate early protein BZLF1.

Raver Ryan M RM Panfil Amanda R AR Hagemeier Stacy R SR Kenney Shannon C SC

Journal of virology 20130515 14

The latent-to-lytic switch of Epstein-Barr virus (EBV) is mediated by the immediate early protein BZLF1 (Z). However, the cellular factors regulating this process remain incompletely characterized. In this report, we show that the B-cell-specific transcription factor Pax5 helps to promote viral latency in B cells by blocking Z function. Although Z was previously shown to directly interact with Pax5 and inhibit its activity, the effect of Pax5 on Z function has not been investigated. Here, we dem ...[more]

PMID: 23678172

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Project description:The Epstein-Barr virus (EBV) BHLF1 gene encodes an abundant linear and several circular RNAs believed to perform noncoding functions during virus replication, although an open reading frame (ORF) is retained among an unknown percentage of EBV isolates. Evidence suggests that BHLF1 is also transcribed during latent infection, which prompted us to investigate the contribution of this locus to latency. Analysis of transcripts transiting BHLF1 revealed that its transcription is widespread among B-cell lines supporting the latency I or III program of EBV protein expression and is more complex than originally presumed. EBV-negative Burkitt lymphoma cell lines infected with either wild-type or two different BHLF1 mutant EBVs were initially indistinguishable in supporting latency III. However, cells infected with BHLF1- virus ultimately transitioned to the more restrictive latency I program, whereas cells infected with wild-type virus either sustained latency III or transitioned more slowly to latency I. Upon infection of primary B cells, which require latency III for growth in vitro, both BHLF1- viruses exhibited variably reduced immortalization potential relative to the wild-type virus. Finally, in transfection experiments, efficient protein expression from an intact BHLF1 ORF required the EBV posttranscriptional regulator protein SM, whose expression is limited to the replicative cycle. Thus, one way in which BHLF1 may contribute to latency is through a mechanism, possibly mediated or regulated by a long noncoding RNA, that supports latency III critical for the establishment of EBV latency and lifelong persistence within its host, whereas any retained protein-dependent function of BHLF1 may be restricted to the replication cycle.IMPORTANCE Epstein-Barr virus (EBV) has significant oncogenic potential that is linked to its latent infection of B lymphocytes, during which virus replication is not supported. The establishment of latent infection, which is lifelong and can precede tumor development by years, requires the concerted actions of nearly a dozen EBV proteins and numerous small non-protein-coding RNAs. Elucidating how these EBV products contribute to latency is crucial for understanding EBV's role in specific malignancies and, ultimately, for clinical intervention. Historically, EBV genes that contribute to virus replication have been excluded from consideration of a role in latency, primarily because of the general incompatibility between virus production and cell survival. However, here, we provide evidence that the genetic locus containing one such gene, BHLF1, indeed contributes to key aspects of EBV latency, including its ability to promote the continuous growth of B lymphocytes, thus providing significant new insight into EBV biology and oncogenic potential.

Project description:Deciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral gene expression and host gene expression. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of immune-suppressed individuals. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs), serving as a model of these lymphomas. In previous studies, reports from our laboratory have described a temporal model for immortalization with an initial phase characterized by expression of Epstein-Barr nuclear antigens (EBNAs), high levels of c-Myc activity, and hyperproliferation in the absence of the latent membrane proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly NFκB-activating LMP1, defining latency III. However, LCLs express a broad distribution of LMP1 such that a subset of these cells express LMP1 at levels similar to those seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA sequencing (mRNA-Seq) on early EBV-infected latency IIb cells and latency III LCLs sorted by NFκB activity. We found that latency IIb transcriptomes clustered independently from latency III independently of NFκB. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-fluorescence in situ hybridization (RNA-FISH) targeting EBV EBNA2 or LMP1 and human CCR7 or MGST1 This report defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.IMPORTANCE EBV is a ubiquitous pathogen, with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single-cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction of the cells express membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely, making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more-efficacious therapies in the future.

Project description:The EBNA1 protein of Epstein-Barr virus (EBV) plays multiple roles in EBV latent infection, including altering cellular pathways relevant for cancer. Here we used microRNA (miRNA) cloning coupled with high-throughput sequencing to identify the effects of EBNA1 on cellular miRNAs in two nasopharyngeal carcinoma cell lines. EBNA1 affected a small percentage of cellular miRNAs in both cell lines, in particular, upregulating multiple let-7 family miRNAs, including let-7a. The effects of EBNA1 on let-7a were verified by demonstrating that EBNA1 silencing in multiple EBV-positive carcinomas downregulated let-7a. Accordingly, the let-7a target, Dicer, was found to be partially downregulated by EBNA1 expression (at the mRNA and protein levels) and upregulated by EBNA1 silencing in EBV-positive cells. Reporter assays based on the Dicer 3' untranslated region with and without let-7a target sites indicated that the effects of EBNA1 on Dicer were mediated by let-7a. EBNA1 was also found to induce the expression of let-7a primary RNAs in a manner dependent on the EBNA1 transcriptional activation region, suggesting that EBNA1 induces let-7a by transactivating the expression of its primary transcripts. Consistent with previous reports that Dicer promotes EBV reactivation, we found that a let-7a mimic inhibited EBV reactivation to the lytic cycle, while a let-7 sponge increased reactivation. The results provide a mechanism by which EBNA1 could promote EBV latency by inducing let-7 miRNAs.The EBNA1 protein of Epstein-Barr virus (EBV) contributes in multiple ways to the latent mode of EBV infection that leads to lifelong infection. In this study, we identify a mechanism by which EBNA1 helps to maintain EBV infection in a latent state. This involves induction of a family of microRNAs (let-7 miRNAs) that in turn decreases the level of the cellular protein Dicer. We demonstrate that let-7 miRNAs inhibit the reactivation of latent EBV, providing an explanation for our previous observation that EBNA1 promotes latency. In addition, since decreased levels of Dicer have been associated with metastatic potential, EBNA1 may increase metastases by downregulating Dicer.

Project description:Epstein-Barr virus (EBV) switches between latent and lytic phases in hosts, which is important in the development of related diseases. However, the underlying mechanism of controlling the viral biphasic life cycle and how EBV mediates this regulation remain largely unknown. This study identified bromodomain-containing protein 7 (BRD7) as a crucial host protein in EBV latent infection. Based on the chromatin immunoprecipitation (ChIP) sequencing of endogenous BRD7 in Burkitt lymphoma cells, we found that EBV drove BRD7 to regulate cellular and viral genomic loci, including the transcriptional activation of c-Myc, a recently reported regulator of EBV latency. Additionally, EBV-mediated BRD7 signals were enriched around the FUSE (far-upstream sequence element) site in chromosome 8 and the enhancer LOC108348026 in the lgH locus, which might activate the c-Myc alleles. Mechanically, EBV-encoded nuclear antigen 1 (EBNA1) bound to BRD7 and colocalized at promoter regions of the related genes, thus serving as cofactors for the maintenance of viral latency. Moreover, the disruption of BRD7 decreased the c-Myc expression, induced the BZLF1 expression, and reactivated the lytic cycle. Our findings reveal the unique role of BRD7 to synergize with EBV in maintaining the viral latency state via chromatin remodeling. This study paves the way for understanding the new molecular mechanism of EBV-induced chromatin remodeling and latent-lytic switch, providing novel therapeutic candidate targets for EBV persistent infection. IMPORTANCE When establishing persistent infection in most human hosts, EBV is usually latent. How the viral latency is maintained in cells remains largely unknown. c-Myc was recently reported to act as a controller of the lytic switch, while whether and how EBV regulates it remain to be explored. Here, we identified that BRD7 is involved in controlling EBV latency. We found that EBV-mediated BRD7 was enriched in both the normal promoter regions and the translocation alleles of c-Myc, and disruption of BRD7 decreased c-Myc expression to reactivate the lytic cycle. We also demonstrated that EBV-encoded EBNA1 bound to and regulated BRD7. Therefore, we reveal a novel mechanism by which EBV can regulate its infection state by coordinating with host BRD7 to target c-Myc. Our findings will help future therapeutic intervention strategies for EBV infection and pathogenesis.

Dataset Information

The B-cell-specific transcription factor and master regulator Pax5 promotes Epstein-Barr virus latency by negatively regulating the viral immediate early protein BZLF1.

Publications

The B-cell-specific transcription factor and master regulator Pax5 promotes Epstein-Barr virus latency by negatively regulating the viral immediate early protein BZLF1.

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