CHARACTERIZATION OF THE EPSTEIN-BARR-VIRUS FP PROMOTER

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) protei n is mediated by the virus Fp promoter in Burkitt lymphoma and nasopha ryngeal carcinoma. This promoter is silent in latently infected a lymp hoblastoid and most Burkitt lymphoma-derived cell lines in vitro, whic h utilize separate promoters approximately 50 kb upstream of Fp to exp ress EBNA proteins. Fp-mediated activation of EBNA-1 expression is als o activated upon induction of the virus replication cycle. We previous ly demonstrated that activation of Fp in Burkitt cells requires cis-re gulatory elements downstream of the site of transcription initiation. We have now mapped two positive regulatory elements within the Fp prom oter. One element contains two potential binding sites for the cellula r transcription factor LBP-1 between +138 and +150. A second regulator y element was mapped between +177 and +192 and can be specifically bou nd in vitro by protein from nuclear extracts of Burkitt cells. Althoug h this element overlaps two partial E2F binding sites and Fp reporter plasmids could be activated in trans by the adenovirus E1A protein in cotransfection experiments, mutational analysis and DNA binding studie s suggest that these are unlikely to be functional E2F response elemen ts within Fp. We also demonstrate that Fp-directed transcription initi ates at multiple sites within both the genome and the Fp reporter plas mids. However, the principal site of transcription initiation within t he genome is not utilized within reporter plasmids, in which the major ity of transcripts initiate at multiple sites between +150 and +200. T his finding suggests that additional elements may be necessary for Fp to function normally in these assays or that the context of Fp within the viral genome is critical to its regulation. (C) 1995 Academic Pres s, Inc.