FUCTIONS OF EPSTEIN BARR VIRUS NUCLEAR ANTIGEN 1

EB病毒（Epstein-Barr virus，EBV）是一种与多种恶性肿瘤相关的疱疹病毒。 EBV的生命周期和发病机制的一个中心方面是潜伏的感染状态，其中病毒染色体作为附加体维持。 单个EBV编码的蛋白EBNA-1允许EBV染色体通过结合oriP而得以维持，其中它指导DNA复制起始，并且其中它还起到防止EBV染色体从有丝分裂活性细胞中丢失的作用。 oriP的复制功能似乎是感染原代B细胞所必需的，尽管当EBV感染培养物中建立的细胞系时不需要oriP。 分子和遗传学研究提出了调查EBNA-1和细胞因子如何指导复制起始于oriP。我们描述了四个具体的目标：（1）我们有证据表明，为了支持复制起始，EBNA-1的两个二聚体必须在oriP形成一个精确的结构，涉及一个大的DNA弯曲。 我们将研究弯曲的角度以及是否需要特定的几何形状。 (2)我们将利用酵母和非洲爪蟾研究中出现的大量关于DNA复制的信息，开始研究可能控制oriP DNA复制起始的人类蛋白质，使用染色质免疫沉淀和体内足迹分析。 (3)我们将分离EBNA-1的复制缺陷突变体，这些突变体保留所有其他EBNA-1活性，因此可能无法执行起始步骤，例如与宿主因子相互作用。 (4)我们将通过绘制新生链的5'端来精确地确定复制在oriP起始的位置。 这些独立且互补的方法中的每一种都应该提供有关复制如何在oriP启动的关键见解。 这些研究也将有助于我们理解人类染色体的复制。

Epstein-Barr virus (EBV) is a herpesvirus which is associated with several malignancies. A central aspect of the life cycle and pathogenesis of EBV is a latent state of infection in which the viral chromosome is maintained as an episome. A single EBV- encoded protein, EBNA-1, allows the EBV chromosome to be maintained by binding to oriP, where it directs DNA replication to initiate and where it also acts to prevent the EBV chromosome from being lost from mitotically active cells. The replication function of oriP appears to be essential for the infection of primary B cells, although it is not needed when EBV infects established cell lines in culture. Molecular and genetic studies are proposed to investigate how EBNA-1 and cellular factors direct replication to initiate at oriP. Four specific aims are described: (1) We have evidence that in order to support replication initiation, two dimers of EBNA-1 must form a precise structure at oriP that involves a large DNA bend. We will investigate the angle of bending and whether a specific geometry is required. (2) We will take advantage of a wealth of information about DNA replication that has emerged from studies of yeast and Xenopus to begin to investigate the human proteins that may control initiation of DNA replication at oriP, using chromatin immunopreciptation and in vivo footprinting assays. (3) We will isolate replication-defective mutants of EBNA-1 that retain all other EBNA-1 activities and, therefore, may fail to perform an initiation step, such as interact with a host factor. (4) We will determine precisely where replication initiates at oriP by mapping the 5' ends of leading nascent strands. Each of these independent and complementary approaches should provide key insight into how replication initiates at oriP. The studies will also to be relevant to our understanding the replication of human chromosomes.