Epstein–Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kB pathway

The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substan15 tially contributes to EBV’s oncogenic potential by activating nuclear factor-iB (NF-iB). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in 20 EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBVnegative B cells. We analyzed two putative NF-iB binding sites in the miR-155 promoter; both sites 25 recruited NF-iB complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-iB sites and with miR-155 overexpression. The 30 induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-iB binding sites was no longer activated 35 by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible 40 miR target.