FUCTIONS OF EPSTEIN BARR VIRUS NUCLEAR ANTIGEN 1

爱泼斯坦-巴尔病毒（EBV）是一种与几种恶性肿瘤相关的疱疹病毒。EBV生命周期和发病机制的一个核心方面是感染的潜伏状态，在这种状态下，病毒染色体被维持为一个插曲。EBV编码的单一蛋白EBNA-1通过与oriP结合来维持EBV染色体，在oriP上，它指导DNA复制的启动，并在oriP上起作用，防止EBV染色体从有丝分裂活跃的细胞中丢失。oriP的复制功能似乎对原代B细胞的感染至关重要，尽管EBV在培养中感染已建立的细胞系时不需要它。分子和遗传学的研究将探讨EBNA-1和细胞因子如何引导复制在oriP上启动。本文描述了四个具体目标：(1)我们有证据表明，为了支持复制起始，ena -1的两个二聚体必须在oriP上形成一个精确的结构，该结构涉及一个大的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)我们将利用从酵母和爪蟾的研究中获得的关于DNA复制的丰富信息，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测定。(3)我们将分离EBNA-1复制缺陷突变体，这些突变体保留了所有其他EBNA-1活性，因此可能无法执行启动步骤，例如与宿主因子相互作用。(4)我们将通过绘制前导新生链的5'端来精确确定oriP上的复制起始位置。这些独立和互补的方法中的每一种都应该提供了解复制如何在oriP上启动的关键见解。这些研究也将与我们对人类染色体复制的理解有关。

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.