Dataset Information

Epstein-Barr virus EBNA1 protein regulates viral latency through effects on let-7 microRNA and dicer.

ABSTRACT: 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.

SUBMITTER: Mansouri S

PROVIDER: S-EPMC4178782 | biostudies-other | 2014 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Epstein-Barr virus EBNA1 protein regulates viral latency through effects on let-7 microRNA and dicer.

Mansouri Sheila S Pan Qun Q Blencowe Benjamin J BJ Claycomb Julie M JM Frappier Lori L

Journal of virology 20140716 19

<h4>Unlabelled</h4>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 effe ...[more]

PMID: 25031339

Similar Datasets

Project description:Epstein-Barr virus (EBV) is a human herpesvirus associated with various tumors. Rather than going through the lytic cycle, EBV maintains latency by limiting the expression of viral genes in tumors. Viral microRNAs (miRNAs) of some herpesviruses have been reported to directly target immediate early genes and suppress lytic induction. In this study, we investigated whether BamHI-A rightward transcript (BART) miRNAs targeted two EBV immediate early genes, BZLF1 and BRLF1. Bioinformatic analysis predicted that 12 different BART miRNAs would target BRLF1. Of these, the results of a luciferase reporter assay indicated that only one interacted with the 3' untranslated region (UTR) of BRLF1: miR-BART20-5p. miR-BART20-5p's effect on gene expression involved two putative seed match sites in the BRLF1 3' UTR, but a mutant version of the miRNA, miR-BART20-5pm, had no effect on expression. As expected from the fact that the entire 3' UTR of BZLF1 resides within the 3' UTR of BRLF1, miR-BART20-5p interacted with the 3' UTR of BZLF1 as well. BZLF1 and BRLF1 mRNA and protein expression was suppressed in cells of an AGS cell line infected with the recombinant Akata strain of EBV (AGS-EBV) transfected with a miR-BART20-5p mimic. The expression of various EBV early proteins was also suppressed by the miR-BART20-5p mimic. In contrast, BZLF1 and BRLF1 expression in AGS-EBV cells transfected with a miR-BART20-5p inhibitor was enhanced. Furthermore, progeny virus production was suppressed by the miR-BART20-5p mimic and enhanced by the miR-BART20-5p inhibitor in AGS-EBV cells induced for the lytic cycle. Our data suggest that miR-BART20-5p plays a key role in latency maintenance in EBV-associated tumors by directly targeting immediate early genes.Herpesviruses maintain latency using various mechanisms and establish lifelong infection in the host. From time to time, herpesviruses are reactivated and express immediate early genes which trigger a lytic cascade, leading to the production of progeny viruses. Recently, some herpesviruses have been shown to use their own microRNAs (miRNAs) to downregulate immediate early genes to inhibit the lytic cycle. This study presents evidence that EBV also downregulates two immediate early genes by miR-BART20-5p to suppress the lytic cycle and progeny virus production. Overall, this is the first study to report the direct regulation of EBV immediate early genes by an EBV miRNA, implying its likely importance in latency maintenance in EBV-associated tumors.

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.

Dataset Information

Epstein-Barr virus EBNA1 protein regulates viral latency through effects on let-7 microRNA and dicer.

Publications

Epstein-Barr virus EBNA1 protein regulates viral latency through effects on let-7 microRNA and dicer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets