DNA/PROTEIN INTERACTION IN HERPES VIRUSES

疱疹病毒是大的DNA病毒，其中几种感染人类细胞，导致无数疾病，有些严重。与这些病毒作斗争需要更多地了解它们的生命周期。DNA复制的起始是抗病毒方法的一个有吸引力的靶点，因为它代表了新病毒产生的最早阶段。单纯疱疹病毒I型（HSV-I）感染人类细胞，并且是最好理解的疱疹病毒。在该项目中，努力将继续集中于病毒和宿主蛋白在HSV-1复制起始的第一阶段中的作用，特别是结合HSV-1起点并充当解旋酶的UL 9蛋白，以及一般单链DNA结合蛋白ICP 8。一个主要的重点将是进一步表征ICP 8和UL 9的相互作用，因为它们打开和解开HSV-1的起源。结合生物化学和电子显微镜（EM）研究的分析将提供详细的机制，包括拓扑异构酶I如何驱动解旋反应以及它是否直接与UL 9结合。这是可能的，宿主细胞热休克（伴侣）蛋白帮助加载UL 9的起源和这种可能性将被探索。这些研究将采用EM，生物化学测定和表面等离子体共振测量来确定在起源处形成并启动复制的蛋白质复合物的性质和结构。使用质粒DNA-蛋白质复合物，其中起始点被UL 9和ICP 8部分解绕，将加入来自HSV-1感染的人细胞的提取物和在昆虫细胞中产生的HSV-1复制体，以了解更多关于复制的后续步骤。长期目标是使用质粒模板在体外重建完整的HSV-1复制。潜伏HSV-I的激活和从潜伏基因组开始复制可能需要从起点去除核小体。GRE（糖皮质激素反应元件）最近已经在HSV-1起源中被鉴定为oriL，并且这可以用于将核小体独特地定位在oriL中的UL 9结合位点上，从而产生分子的、对糖皮质激素敏感的开关。这将使用体外染色质组装和足迹法进行检测。高分辨电镜将用于确定几种HSV-1 DNA-蛋白质复合物的精细结构。一个与肯尼博士的合作项目将探讨EB病毒系统中的许多相同问题。

The Herpes viruses are large DNA viruses several of which infect human cells to cause a myriad of diseases some, severe. Combating these viruses will require knowing more about their life cycles. The initiation of DNA replication presents an attractive target for anti-viral approaches as it represents the earliest stage in the production of new virus. Herpes Simplex type l (HSV- l) infects human cells and is the best understood of the Herpes viruses. In this project, efforts will continue to focus on the action of viral and host proteins in the first stages of initiation of HSV- l replication, in particular UL9 protein which binds to the HSV-l origins and acts as a helicase, and ICP8, the general single strand DNA binding protein. A major focus will be on further characterizing the interactions of ICP8 and UL9 as they open and unwind the HSV-l origin. Analysis combining biochemical and electron microscopic (EM) studies will provide a detailed mechanism including how topoisomerase I drives the unwinding reaction and whether it binds directly to UL9. It is possible that host cell heat shock (chaperone) proteins help load UL9 onto the origins and this possibility will be explored. These studies will employ EM, biochemical assays, and surface plasmon resonance measurements to define the nature and structure of the protein complexes that form at the origins and initiate replication. Using plasmid DNA-protein complexes in which the origin is partially unwound by UL9 and ICP8, extracts from HSV-1 infected human cells and an HSV-l replisome generated in insect cells will be added to learn more about the subsequent steps of replication. The long-range goal is to reconstitute full HSV- l replication using plasmid templates in vitro. Activation of latent HSV- l and initiation of replication from latent genomes likely requires removal of nucleosomes from the origin. A GRE (glucocorticoid response element) has recently been identified in the HSV-l origin termed oriL and this may act to uniquely position nucleosomes over the UL9 binding sites in oriL, creating a molecular, hormone-sensitive switch. This will be tested using in vitro chromatin assembly and footprinting methods. High resolution EM will be utilized to determine the fine structure of several HSV- l DNA-protein complexes. A collaborative project with Dr. Kenney of this program project will explore many of these same questions in the EBV system.