Epstein Barr virus (EBV) infection is associated with the development of post-transplant lymphoproliferative disorder (PTLD) which can manifest as aggressive B cell lymphomas in transplant recipients. It is generally accepted that immunosuppression required to prevent graft rejection impairs host anti-viral immunity, thereby permitting the expansion of EBV-infected B cells in PTLD. Less clear are the viral- and cell-intrinsic factors that drive proliferation of EBV+ B cells. The purpose of this study was to determine whether EBV co-opts the host B cell microRNA (miR) machinery to promote lymphoma expansion. miR are small, non-coding RNA that play an important role in post-transcriptional regulation of cellular gene expression. We focused on miR regulation of cellular IL-10, previously shown to be an autocrine growth factor for EBV+ B cell lymphomas. miR qPCR gene arrays were performed on normal B cells from healthy donors (n=4), lymphoblastoid cell lines (LCL) generated by infecting B cells with the B95.8 strain of EBV (n=4), and EBV+ B cell lymphoma lines isolated from patients with PTLD (n=6). ANOVA analysis identified 133 miR modulated by EBV infection of B cells. Of these 133 miR, nine were predicted to target the 3'UTR of IL-10 by TargetScan and miRanda in silico screen. Only one miR, miR-194, was downregulated uniquely in the PTLD-derived EBV+ B lymphoma cell lines. Overexpression of miR-194 in 293T cells significantly inhibited expression of Renilla luciferase containing the 3'UTR of IL-10, compared to the control vector that did not express miR. In contrast, a vector expressing miR-340, which is modulated by EBV but is not predicted to target the 3'UTR of IL-10, had no effect on Renilla luciferase expression. Lentiviral delivery of miR-194 into EBV+ B cell lymphoma lines from PTLD patients significantly reduced IL-10 production in all four lines tested (p<0.05, range of decrease=43-72%). In contrast, IL-10 production was not altered in cells transduced with control GFP plasmid. Thus, EBV infection rewires the host B cell miR machinery to specifically suppress miR-194 and override control of IL-10 expression, thereby contributing to constitutive production of this autocrine growth factor for B cell lymphomas. Strategies that restore regulation of B cell miR may offer new approaches for treatment of EBV+ PTLD.

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