Epstein-Barr virus (EBV) infection has been associated with the development of several types of human malignancy. The horizontal transmission of EBV from host to host requires activation of the lytic origin of replication, oriLyt. Lytic EBV replication requires six vira1ly encoded replication proteins (BALF5, BMRF1, BALF2, BBLF4, BSLF1, and BBLF2/3). In addition, the immediate-early transactivator protein, BZLF1, is essential for oriLyt replication, even when the other viral replication proteins are expressed by a constitutively active promoter. Although the functional equivalent of the herpes simplex virus origin binding protein, UL9, has not been definitively identified for EBV, only two small regions in the EBV oriLyt (the "upstream" and "downstream" essential domains) are absolutely required for replication. Binding of BZLF1 to the upstream essential domain is necessary for oriLyt replication. We have recently discovered that the downstream essential domain is transcriptionally activated by the BMRF1 protein (the viral polymerase processivity factor), and shown that small mutations in oriLyt which abolish BMRF1-induced transactivation also abolish oriLyt replication. Thus, the BMRF1 protein appears to have two separate roles in oriLyt replication, serving both as the polymerase processivity factor, and a transcriptional activator. In this grant, we propose to study the regulation of oriLyt by the viral proteins, BMRF1 and BZLF1. Our specific aims are to l) define the mechanism by which BMRF1 transcriptionally activates oriLyt, 2) determine if the transcriptional activator function of BMRF1 (apart from its polymerase processivity function) is required for oriLyt replication, 3) examine the effects of BZLF1 and BMRF1 on oriLyt structure using electron microscopy, and 4) determine if EBV (like other herpesviruses) disrupts PML-associated nuclear bodies during lytic infection, and if so, whether this effect is required for oriLyt replication. We hypothesize that transcriptional activation of oriLyt by BMRF1 is required for lytic EBV replication, and that BZLF1 and/or BMRF1 serves as the functional origin- binding protein of oriLyt.