Role and mechanism of action of gamma herpesvirus microRNAs

Duke University, Durham NC

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Abstract

Humans are hosts for two lymphotropic γ-herpesviruses, Epstein-Barr virus (EBV) and Kaposi[unreadable]s sarcomaassociated herpesvirus (KSHV), both of which have the ability to induce cancer, especially in immunocompromised patients. Thus, EBV-associated tumors are far more prevalent in AIDS patients, and EBV-associated non-Hodgkin[unreadable]s lymphomas, particularly diffuse large B-cell lymphomas (DLBCL), are common AIDS-related malignancies. EBV and KSHV also have in common the ability to establish latent infections and, during latency, both viruses express a substantial number of viral microRNAs (miRNAs), many of which were first reported by this laboratory. An important attribute of EBV, which distinguishes EBV from KSHV, is its ability to infect primary B cells in culture to establish a latent infection (latency III) that also induces immortalization of the infected cells. The resultant lymphoblastoid cell lines (LCLs), are similar to DLBCL cells in that they express the same small subset of EBV proteins and multiple virally encoded microRNAs. In addition, EBV infection of B cells dramatically upregulates the expression of several cellular miRNAs, including miR-155, miR-21 and miR-146a, that are also highly expressed in DLBCL. Of note, miR-21 and miR-155 are so-called [unreadable]oncomirs[unreadable], whose expression predisposes cells to oncogenic transformation, while miR-146 functions as a negative regulator of the antiviral interferon response. EBV also expresses multiple viral miRNAs in virally-transformed epithelial cells, including particularly nasopharyngeal carcinoma (NPC) cells, but the contribution of these EBV-encoded miRNAs, as well as virally-induced cellular miRNAs, remains unclear in both virally transformed B cells, including LCLs and DLBCLs, as well as in NPC. In this grant, we propose a series of experiments to define the contribution of these viral and cellular miRNAs to maintaining the growth and viability of these latently EBVinfected, transformed cells. Preliminary data indicate that at least two EBV-encoded miRNAs, as well as cellular miR-155, play key roles in facilitating LCL and DLBCL formation and/or maintenance. We will use cross-linking immunoprecipitation (CLIP) assays, in combination with indicator assays and Western blots, to catalog all of the mRNAs targeted by these viral and cellular miRNAs in EBV-infected LCL, DLBCL and NPC cells. Finally, we will use RNA interference and gene complementation to determine which mRNA targets for these phenotypically important miRNAs actually confer the observed growth advantage when downregulated. Our initial results indicate that the miRNAs expressed during latency by EBV and KHSV, despite their lack of sequence homology, nevertheless target a number of identical host mRNAs. This program of research is designed to lead to a mechanistic understanding of how virally encoded and virally induced cellular miRNAs contribute to the induction and maintenance of cellular immortalization/transformation and has the potential to lead to new approaches to the treatment of the EBV-induced AIDS-associated malignancies.