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Epstein-Barr virus BamHI W repeat number limits EBNA2/EBNA-LP coexpression in newly infected B cells and the efficiency of B-cell transformation: a rationale for the multiple W repeats in wild-type virus strains.

ABSTRACT: The genome of Epstein-Barr virus (EBV), a gammaherpesvirus with potent B-cell growth-transforming ability, contains multiple copies of a 3-kb BamHI W repeat sequence; each repeat carries (i) a promoter (Wp) that initiates transformation by driving EBNA-LP and EBNA2 expression and (ii) the W1W2 exons encoding the functionally active repeat domain of EBNA-LP. The W repeat copy number of a virus therefore influences two potential determinants of its transforming ability: the number of available Wp copies and the maximum size of the encoded EBNA-LP. Here, using recombinant EBVs, we show that optimal B-cell transformation requires a minimum of 5 W repeats (5W); the levels of transforming ability fall progressively with viruses carrying 4, 3, and 2 W repeats, as do the levels of Wp-initiated transcripts expressed early postinfection (p.i.), while viruses with 1 copy of the wild-type W repeat (1W) and 0W are completely nontransforming. We therefore suggest that genetic analyses of EBV transforming function should ensure that wild-type and mutant strains have equal numbers (ideally at least 5) of W copies if the analysis is not to be compromised. Attempts to enhance the transforming function of low-W-copy-number viruses, via the activity of helper EBV strains or by gene repair, suggested that the critical defect is not related to EBNA-LP size but to the failure to achieve sufficiently strong coexpression of EBNA-LP and EBNA2 early postinfection. We further show by the results of ex vivo assays that EBV strains in the blood of infected individuals typically have a mean of 5 to 8 W copies, consistent with the view that evolution has selected for viruses with an optimal transforming function.

SUBMITTER: Tierney RJ

PROVIDER: S-EPMC3209410 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Epstein-Barr virus BamHI W repeat number limits EBNA2/EBNA-LP coexpression in newly infected B cells and the efficiency of B-cell transformation: a rationale for the multiple W repeats in wild-type virus strains.

Tierney Rosemary J RJ Kao Kuan-Yu KY Nagra Jasdeep K JK Rickinson Alan B AB

Journal of virology 20110928 23

The genome of Epstein-Barr virus (EBV), a gammaherpesvirus with potent B-cell growth-transforming ability, contains multiple copies of a 3-kb BamHI W repeat sequence; each repeat carries (i) a promoter (Wp) that initiates transformation by driving EBNA-LP and EBNA2 expression and (ii) the W1W2 exons encoding the functionally active repeat domain of EBNA-LP. The W repeat copy number of a virus therefore influences two potential determinants of its transforming ability: the number of available Wp ...[more]

PMID: 21957300

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Project description:Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knock Out (LPKO) virus-infected cells express EBNA2-activated genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-γ coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging cellular transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data and confirmed their biochemical association in LCLs by endogenous co-immunoprecipitation. Moreover, we found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. Finally, we used Cas9 to knockout YY1 in primary total B cells and naïve B cells prior to EBV infection and found YY1 to be essential for EBV-mediated transformation. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells.

Project description:Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knockout (LPKO) virus-infected cells express EBNA2-activated cellular genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-g coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data. By Cut&Run, YY1 peaks unique to WT compared to LPKO LCLs occur at more highly expressed genes. Moreover, Cas9 knockout of YY1 in primary B cells prior to EBV infection indicated YY1 to be important for EBV-mediated transformation. We confirmed EBNA-LP and YY1 biochemical association in LCLs by endogenous co-immunoprecipitation and found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells.

Project description:The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.

Project description:Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knock Out (LPKO) virus-infected cells express EBNA2-activated cellular genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-g coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging cellular transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data. By Cut&Run, YY1 peaks unique to WT compared to LPKO LCLs occur at more highly expressed genes. Moreover, Cas9 knockout of YY1 in primary B cells prior to EBV infection indicted YY1 to be essential for EBV-mediated transformation. We confirmed EBNA-LP and YY1and confirmed their biochemical association in LCLs by endogenous co-immunoprecipitation and. Moreover, we found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. Finally, we used Cas9 to knockout YY1 in primary total B cells and naïve B cells prior to EBV infection and found YY1 to be essential for EBV-mediated transformation. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells.

Dataset Information

Epstein-Barr virus BamHI W repeat number limits EBNA2/EBNA-LP coexpression in newly infected B cells and the efficiency of B-cell transformation: a rationale for the multiple W repeats in wild-type virus strains.

Publications

Epstein-Barr virus BamHI W repeat number limits EBNA2/EBNA-LP coexpression in newly infected B cells and the efficiency of B-cell transformation: a rationale for the multiple W repeats in wild-type virus strains.

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