REGULATION OF EBV ORI-LYT BY CELLULAR AND VIRAL PROTEINS

eb病毒（EBV）感染与几种人类恶性肿瘤的发展有关。EBV从宿主到宿主的水平传播需要激活复制的裂解起源，或ilyt。eb病毒的裂解复制需要6种病毒编码的复制蛋白（BALF5、BMRF1、BALF2、BBLF4、BSLF1和BBLF2/3）。此外，即时早期反激活蛋白BZLF1对oriLyt复制至关重要，即使其他病毒复制蛋白是由组成性活性启动子表达的。虽然在EBV中还没有明确地鉴定出与单纯疱疹病毒起源结合蛋白UL9功能相当的UL9，但在EBV oriLyt中只有两个小区域（“上游”和“下游”基本结构域）是绝对需要复制的。BZLF1与上游必需结构域的结合是oriLyt复制所必需的。我们最近发现下游必需结构域被BMRF1蛋白（病毒聚合酶加工因子）转录激活，并表明oriLyt中的小突变可以消除BMRF1诱导的转激活，也可以消除oriLyt复制。因此，BMRF1蛋白在oriLyt复制中似乎有两种独立的作用，既作为聚合酶加工因子，又作为转录激活因子。本课题拟研究病毒蛋白BMRF1和BZLF1对oriLyt的调控作用。我们的具体目标是1)确定BMRF1转录激活oriLyt的机制，2)确定BMRF1转录激活因子的功能（除了其聚合酶加工功能）是否需要oriLyt复制，3)使用电子显微镜检查BZLF1和BMRF1对oriLyt结构的影响，以及4)确定EBV（像其他疱疹病毒一样）是否在裂解感染期间破坏pml相关的核体，如果是这样，oriLyt复制是否需要这种效果。我们假设BMRF1对oriLyt的转录激活是裂解EBV复制所必需的，并且BZLF1和/或BMRF1是oriLyt的功能性起源结合蛋白。

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.