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

The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially 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 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 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 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 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 miR target. INTRODUCTION Epstein–Barr virus (EBV), which infects over 90% of the adults, appears to have evolved to exploit the normal biology of B-cell development in order to persist as a life-long asymptomatic infection. However, EBV can contribute to oncogenesis. Indeed, it is frequently found in Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma and lymphoproliferative diseases in immunesuppressed individuals (1,2). EBV-related oncogenesis is principally associated with latency during which only a limited subset of the full repertoire of viral genes are transcribed. In addition to protein-coding genes, the noncoding RNAs EBER1, EBER2 and a set of EBV-encoded microRNAs (miRNAs) are expressed in all three forms of latency (2). Of the genes expressed during viral latency in EBV-associated diseases, LMP1 is the one most implicated in tumor formation (3,4). LMP1 is invariably expressed in Burkitt’s lymphoma, it is required for EBVmediated transformation of lymphocytes in vitro and it transforms rodent fibroblasts. Moreover, transgenic mice expressing LMP1 under the control of an immunoglobulin promoter develop B lymphocyte tumors more frequently than wild-type mice (3). LMP1 is a six-transmembrane constitutively active signaling molecule that functionally mimics members of the cellular tumor necrosis factor receptor (TNFR) family. While the transmembrane domains are required for aggregation and constitutive activation of LMP1 (5–8), two cytoplasmic domains, i.e. the C-terminal activator regions 1 and 2 (CTAR-1 and -2), are critical for the transforming properties of LMP1 (9–12). Together, these signaling domains act through cellular TNFR-associated factors The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors *To whom correspondence should be addressed. Tel: +39 081 7463627; Fax: +39 081 7463205; Email: mallardo@dbbm.unina.it Present address: Sven Kroening, Universitätsklinikum Erlangen, Medizinische Klinik 4, Erlagen, DE, Germany