AN ATF CRE ELEMENT MEDIATES BOTH EBNA2-DEPENDENT AND EBNA2-INDEPENDENT ACTIVATION OF THE EPSTEIN-BARR-VIRUS LMP1 GENE PROMOTER/

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a vir al oncogene whose expression is regulated by both viral and cellular f actors. EBV nuclear antigen 2 (EBNA2) is a potent transactivator of LM P1 expression in human B cells, and several EBNA2 response elements ha ve been identified in the promoter regulatory sequence (LRS). We have previously shown that an activating transcription factor/cyclic AMP re sponse element (ATF/CRE) site in LRS is involved in EBNA2 responsivene ss. We now establish the importance of the ATF/CRE element by mutation al analysis and show that both EBNA2-dependent activation and EBNA2-in dependent activation of the promoter occur via this site but are media ted by separate sets of factors. An electrophoretic mobility shift ass ay (EMSA) with specific antibodies showed that the ATF-1, CREB-1, ATF- 2 and c-Jun factors bind to the site as ATF-1/CREB-1 and ATF-2/c-Jun h eterodimers whereas the Spl and Sp3 factors bind to an adjacent Sp sit e. Overexpression of ATF-1 and CREB-1 in the cells by expression vecto rs demonstrated that homodimeric as well as heterodimeric forms of the factors transactivate the LMP1 promoter in an EBNA2-independent manne r. The homodimers of ATF-2 and c-Jun did not significantly stimulate p romoter activity. In contrast, the ATF-2/c-Jun heterodimer had only a minor stimulatory effect in the absence of EBNA2 but induced a strong transactivation of the LMP1 promoter when coexpressed with this protei n. Evidence for a direct interaction between the ATF-2/c-Jun heterodim eric complex and EBNA2 was obtained by EMSA and coimmunoprecipitation experiments. Thus, our results suggest that EBNA2-induced transactivat ion via the ATF/CRE site occurs through a direct contact between EBNA2 and an ATF-2/c-Jun heterodimer. EBNA2-independent promoter activation via this site, on the other hand, is mediated by a heterodimeric comp lex between the ATF-1 and CREB-1 factors.