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Trans-Repression of protein expression dependent on the Epstein-Barr virus promoter Wp during latency.

ABSTRACT: An ordered silencing of Epstein-Barr virus (EBV) latency gene transcription is critical for establishment of persistent infection within B lymphocytes, yet the mechanisms responsible and the role that the virus itself may play are unclear. Here we describe two B-cell superinfection models with which to address these problems. In the first, Burkitt lymphoma (BL) cells that maintain latency I, when superinfected, initially supported transcription from the common EBNA promoters Wp and Cp (latency III) but ultimately transitioned to latency I (Cp/Wp silent), an essential requirement for establishment of EBV latency in vivo. We used this model to test whether the early lytic-cycle gene BHLF1, implicated in silencing of the Cp/Wp locus, is required to establish latency I. Upon superinfection with EBV deleted for the BHLF1 locus, however, we have demonstrated that BHLF1 is not essential for this aspect of EBV latency. In the second model, BL cells that maintain Wp-restricted latency, a variant program in which Cp is silent but Wp remains active, sustained the latency III program of transcription from the superinfecting-virus genomes, failing to transition to latency I. Importantly, there was substantial reduction in Wp-mediated protein expression from endogenous EBV genomes, in the absence of Cp reactivation, that could occur independent of a parallel decrease in mRNA. Thus, our data provide evidence of a novel, potentially posttranscriptional mechanism for trans-repression of Wp-dependent gene expression. We suggest that this may ensure against overexpression of the EBV nuclear antigens (EBNAs) prior to the transcriptional repression of Wp in cis that occurs upon activation of Cp.

SUBMITTER: Hughes DJ

PROVIDER: S-EPMC3194970 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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trans-Repression of protein expression dependent on the Epstein-Barr virus promoter Wp during latency.

Hughes David J DJ Dickerson Carol A CA Shaner Marie S MS Sample Clare E CE Sample Jeffery T JT

Journal of virology 20110824 21

An ordered silencing of Epstein-Barr virus (EBV) latency gene transcription is critical for establishment of persistent infection within B lymphocytes, yet the mechanisms responsible and the role that the virus itself may play are unclear. Here we describe two B-cell superinfection models with which to address these problems. In the first, Burkitt lymphoma (BL) cells that maintain latency I, when superinfected, initially supported transcription from the common EBNA promoters Wp and Cp (latency I ...[more]

PMID: 21865378

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Project description:UnlabelledHuman B cells, the main target of Epstein-Barr virus (EBV), can display several types of latent viral protein expression, denoted 0, I, IIa, IIb, or III. Of these, only type III expression induces proliferation of cells in vitro. These latency types are present at specific stages of infection and are also characteristic of different tumor types, but their generation is not fully understood. In this study, we analyzed the role of T cells in the regulation of EBV viral latency by using humanized NOD/SCID/IL2Rγ(-/-) mice. Several spleens presented macroscopic tumors 4 weeks after infection. Explanted spleen B cells from some of the EBV-infected mice proliferated in vitro, but this was usually lowered when cyclosporine was added to the cultures. This suggested that the in vitro growth of EBV-infected B cells required T cell help; thus, cells other than type III cells were also present in the spleens. Quantitative PCR analysis of promoter activities specific for the different EBV latency types confirmed that in addition to type III cells, type IIa and type I cells were present in the spleen. The relative usage of the viral promoter specific for I and IIa latency types (Q promoter) was higher in CD8(+) cell-depleted mice, and it was absent from CD4(+) cell-depleted mice. These results indicate that CD4(+) T cells are necessary for the generation/maintenance of cells with latency I/IIa in the humanized mice. CD4(+) T cells contributed to this process through their CD40L expression.ImportanceAt primary infection with EBV, the infected B cells are proliferating and express viral proteins that have transforming potential. However, when the acute infection is resolved, in healthy individuals EBV is carried by a small fraction of B cells that express a restricted number of viral proteins unable to induce proliferation. Understanding the details of this transition is of fundamental importance. We studied this question in humanized mice by manipulating their different T cell compartments before and during infection with EBV. Our results indicate that CD4(+) T cells are responsible for the switch to a nonproliferating EBV program during primary infection with EBV.

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.

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Trans-Repression of protein expression dependent on the Epstein-Barr virus promoter Wp during latency.

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trans-Repression of protein expression dependent on the Epstein-Barr virus promoter Wp during latency.

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