Epstein-Barr virus nuclear antigen 3C putative repression domain mediates coactivation of the LMP1 promoter with EBNA-2

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) regulates virus a nd cell genes and is essential for EBV-mediated transformation of primary B lymphocytes. EBNA-3C associates with the cellular DNA sequence-specific tr anscription factors RBP-J kappa and PU.1 and coactivates the EBV LMP1 promo ter with EBNA-2 in BL2 and Raji cells under conditions of restrictive growt h. We now find that EBNA-3C is similar to EBNA-LP in coactivating the LMP1 promoter with EBNA-2 in non-EBV-infected Burkitt lymphoma cells under condi tions of maximal cell growth, whereas the EBV Cp promoter is repressed unde r the same conditions. EBNA-3A and EBNA-3B coactivation are at most 40% tha t of EBNA-3C. The RBP-J kappa binding sites of EBNA-2 and the LMP1 promoter are not required for EBNA-3C coactivation, whereas the PU.1 site in the LM P1 promoter is required for EBNA-2-mediated activation and EBNA-3C coactiva tion. EBNA-3C amino acids (aa) 365 to 545, including most of the previously identified repression domain (M. Bain, R. J. Watson, P. J. Farrell, and M. J. Allday. J. Virol. 70:2481-2489, 1996), are necessary and sufficient for coactivation with wild-type EBNA-2. EBNA-3C can also coactivate with the E BNA-2 acidic activating domain; this activation does not require aa 343 to 545. These data indicate that there are at least two mechanisms by which EB NA-3C coactivates the LMP1 promoter with EBNA-2. Of the proteins that inter act with EBNA-3C in a yeast two-hybrid screen, only the ubiquitin-like prot eins SUMO-1 and SUMO-3/hSMT3B map to aa 365 to 545, implicating these molec ules in EBNA-3C coactivation. In addition, SUMO-1 associates at a high leve l with EBNA-3C in lymphoblasts. Promoter coactivation by EBNA-3C is likely to be important in ensuring adequate levels of LMP1, while inhibition of th e EBNA-Cp promoter under the same conditions prevents uncontrolled upregula tion of EBNA expression from a positive-feedback loop.