DNA/PROTEIN INTERACTION IN HERPES VIRUSES

疱疹病毒是一种大型DNA病毒，其中几种会感染人类细胞，导致无数疾病，有些是严重的。抗击这些病毒需要更多地了解它们的生命周期。DNA复制的启动是抗病毒方法的一个有吸引力的目标，因为它代表了新病毒产生的最早阶段。单纯疱疹病毒L(HSV-L)感染人类细胞，是对疱疹病毒最了解的病毒。在这个项目中，我们将继续致力于病毒和宿主蛋白在单纯疱疹病毒-L复制启动的第一阶段的作用，特别是与单纯疱疹病毒L起源结合并作为解旋酶的UL9蛋白，以及一般的单链DNA结合蛋白ICP8。一个主要的重点将是进一步表征ICP8和UL9在打开和解开单纯疱疹病毒-L起源时的相互作用。结合生化和电子显微镜(EM)研究的分析将提供详细的机制，包括拓扑异构酶I如何驱动解离反应，以及它是否直接与UL9结合。宿主细胞热休克(伴侣)蛋白可能帮助将UL9加载到起源上，这种可能性将被探索。这些研究将使用EM、生化分析和表面等离子体共振测量来确定在起源形成并启动复制的蛋白质复合体的性质和结构。利用UL9和ICP8部分揭开起始点的质粒DNA-蛋白质复合体，将添加从感染HSV-1的人类细胞中提取的提取物和在昆虫细胞中产生的HSV-L复制体，以了解更多关于后续复制步骤的信息。远期目标是在体外使用质粒模板重建完整的单纯疱疹病毒-L复制。潜伏的单纯疱疹病毒L的激活和从潜伏的基因组开始复制可能需要从起源上去除核小体。最近在单纯疱疹病毒L的起源中发现了一种糖皮质激素反应元件，称为oriL，它可能作用于将核小体唯一地定位在oriL的UL9结合部位上，从而产生一个分子上的激素敏感开关。这将使用体外染色质组装和足迹方法进行测试。高分辨电子显微镜将用于确定几种单纯疱疹病毒L脱氧核糖核酸-蛋白质复合体的精细结构。这个项目的一个与肯尼博士合作的项目将探索EBV系统中的许多同样的问题。

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.