Epstein-Barr virus (EBV) is a human herpesvirus with a well-established association with human neoplasia EBV is consistently associated with endemic Burkitt's lymphoma, nasopharyngeal carcinoma, post-transplant lymphoma, and primary central nervous system lymphoma in AIDS. A proportion of mixed cellularity Hodgkin's lymphomas, systemic lymphomas in AIDS, nasal T cell lymphomas and undifferentiated gastric carcinomas are also EBV associated. Cotransfection-replication assays have identified six essential EBV replication genes, BALF5 (polymerase), BMRF1 (pol.processivity factor), BALF2(ssDNA binding protein), BSLF1(primase), BBLF4(helicase) and BBLF2/3(primase assoc.protein) whose products are functional homologs of replication proteins found in herpes simplex virus. Unlike HSV (or SV4O or HPV viruses), EBV does not encode a UL9-like origin binding protein that has helicase activity. EBV lytic gene expression is regulated by three viral transactivators, Zta, Rta and Mta. Both Zta and Mta are also required for oriLyt replication in the transient replication assay. The present application seeks: (1) To characterize Mta functionally in order to discriminate between its gene regulation and replication roles. The cis-acting sequences within the replication genes that render them sensitive to Mta transactivation will be determined in transfection assays that compare the effects of introducing different ORF and 5' and 3' untranslated sequences. Mutagenesis of Mta will be undertaken to identify domains required for Mta transactivation and replication functions. The contribution of Mta to the intracellular compartmentalization of the other replication proteins will be examined. (2) To define the contribution of Zta to oriLyt replication. The region of the Zta activation domain required for replication function will be defined by mutagenesis. Potential interactions between Zta and the viral replication proteins will be evaluated using co-immunoprecipitation assays, GST-affinity assays and screening in a directed yeast two-hybrid system. The contribution of Zta to the localization of replication proteins to pre-replicative foci will also be examined. (3) To examine the role of certain cis-acting transcriptional signals in oriLyt Elements that confer TPA-responsiveness on the oriLyt enhancer will be defined by DNase I footprinting and EMSA and their general contribution to lytic cycle permissivity evaluated. The sequences that transcription of the oriLyt promoter will be mapped and the cellular DNA binding negatively regulate factor(s) involved in promoter repression will be characterized.
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