REGULATION OF EBV ORI-LYT BY CELLULAR AND VIRAL PROTEINS

EB病毒（EBV）感染与几种类型的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裂解性复制起点oriLyt。裂解性EBV复制需要六种病毒编码的复制蛋白（BALF 5、BMRF 1、BALF 2、BBLF 4、BSLF 1和BBLF 2/3）。此外，即时早期反式激活蛋白BZLF 1是oriLyt复制所必需的，即使其他病毒复制蛋白由组成型活性启动子表达。虽然单纯疱疹病毒起源结合蛋白UL 9的功能等价物尚未被确定用于EBV，但EBV oriLyt中只有两个小区域（“上游”和“下游”必需结构域）是复制绝对需要的。BZLF 1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，下游的基本结构域是转录激活的BMRF 1蛋白（病毒聚合酶合成因子），并表明，小的突变oriLyt废除BMRF 1诱导的反式激活也废除oriLyt复制。因此，BMRF 1蛋白似乎在oriLyt复制中具有两个独立的作用，既作为聚合酶持续合成因子，又作为转录激活因子。在这项研究中，我们计划研究病毒蛋白BMRF 1和BZLF 1对oriLyt的调控。我们的具体目标是：1）确定BMRF 1转录激活oriLyt的机制，2）确定BMRF 1的转录激活因子功能是否与oriLyt的转录激活因子功能有关。（除了它的聚合酶持续合成功能之外）是oriLyt复制所需的，3）使用电子显微镜检查BZLF 1和BMRF 1对oriLyt结构的影响，和4）确定EBV（像其它疱疹病毒一样）是否在裂解感染期间破坏PML相关的核体，如果是，这种作用是否是oriLyt复制所需的。我们假设BMRF 1对oriLyt的转录激活是裂解性EBV复制所需的，并且BZLF 1和/或BMRF 1充当oriLyt的功能性起始结合蛋白。

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.