Herpes Simplex Virus Capsid Assembly and DNA Packaging

描述（由申请方提供）：人类疱疹病毒感染在人群中流行，某些疱疹病毒可导致严重疾病，尤其是在免疫受损个体和新生儿中。免疫功能低下人群的增加产生了对疱疹病毒的抗病毒药物的未满足的医疗需求。拟议项目的目标是通过分析这一过程所需的两种病毒蛋白UL 25和UL 28，更好地了解单纯疱疹病毒（HSV）中的DNA切割和包装。拟议研究背后的具体假设是，UL 25和UL 28是在切割包装反应的不同阶段将DNA包装到HSV衣壳中所必需的。UL 28蛋白是将多联体DNA切割成单位长度分子所必需的，而UL 25在含有衣壳的DNA的产生中起重要作用。虽然重点将放在UL 25和UL 28上，但目标是使用遗传和生物化学方法来表征切割/包装过程，以检查UL 25和UL 28基因中的突变如何影响这些蛋白质与（i）DNA，（ii）衣壳蛋白，（iii）切割/包装蛋白和（iv）宿主细胞蛋白的相互作用。该项目的具体目标是：1。确定UL 25的衣壳位置、DNA结合特异性和蛋白质-蛋白质相互作用。2.通过鉴定我们分离的一系列HSV-1 UL 28突变体，鉴定UL 28的功能结构域，这些功能结构域对于衣壳掺入、结合病毒DNA包装位点以及与末端酶（UL 15）和门蛋白（UL 6）的相互作用是重要的;并针对致死性UL 28接头之一的回复体定位第二位点突变，插入突变体，因为标记拯救和DNA测序已经证明该突变不定位于UL 28基因。3.使用蛋白质组学和生物化学方法检查UL 25和UL 28与病毒和细胞蛋白的相互作用，以鉴定参与切割/包装反应的基本蛋白质-蛋白质相互作用和蛋白质复合物。这些研究旨在阐明HSV DNA切割和包装的机制，并可能为开发抗病毒药物提供新的靶点。

DESCRIPTION (provided by applicant): Infections with human herpesviruses are endemic in the population with some herpesviruses causing severe disease, especially in immune impaired individuals and newborns. The increased immunocompromised population has created an unmet medical need for antivirals against herpesviruses. The goal of the proposed project is to gain a better understanding of DNA cleavage and packaging in herpes simplex virus (HSV) through the analysis of two viral proteins, UL25 and UL28, that are required for this process. The specific hypothesis behind the proposed research is that UL25 and UL28 are required for packaging DNA into HSV capsids at separate stages in the cleavage packaging reaction. The UL28 protein is required for cleavage of concatemeric DNA into unit length molecules while UL25 serves an essential function in the production of DNA containing capsids. Although, the focus will be on UL25 and UL28 the goal is to characterize the cleavage/packaging process using genetic and biochemical approaches to examine how mutations in the UL25 and UL28 genes affect the interaction of these proteins with: (i) DNA, (ii) capsid proteins, (iii) cleavage/packaging proteins and (iv) host cell proteins. Specific goals of the project are to: 1. Determine the capsid location, DNA binding specificity and protein-protein interactions of UL25. 2. Identify functional domains of UL28 that are important for capsid incorporation, binding viral DNA packaging sites and for interaction with terminase (UL15) and portal protein (UL6) by characterizing a series of HSV-1 UL28 mutants that we have isolated; and map the second site mutation for a revertent of one of the lethal UL28 linker- insertion mutants since marker rescue and DNA sequencing have demonstrated that the mutation does not map to the UL28 gene. 3. Examine the interaction of UL25 and UL28 with viral and cell proteins using proteomic and biochemical approaches in order to identify essential protein-protein interactions and protein complexes that are involved in the cleavage/packaging reaction. These studies aim to elucidate the mechanism underlying HSV DNA cleavage and packaging and may suggest novel targets for the development of antivirals.