Herpes Simplex Virus Capsid Assembly and DNA Packaging

描述(申请人提供)：人类疱疹病毒感染在人群中是地方性的，一些疱疹病毒会导致严重的疾病，特别是在免疫受损的个人和新生儿中。免疫受损人口的增加创造了对抗疱疹病毒药物的未得到满足的医疗需求。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UL25和UL28，更好地了解DNA在单纯疱疹病毒(HSV)中的切割和包装。这项研究背后的具体假设是，在切割包装反应的不同阶段，UL25和UL28是将DNA包装到HSV衣壳中所必需的。UL28蛋白是将连体DNA裂解成单位长度分子所必需的，而UL25蛋白在产生含有衣壳的DNA中起着至关重要的作用。虽然重点将放在UL25和UL28上，但目标是利用遗传和生化方法来表征切割/包装过程，以检查UL25和UL28基因的突变如何影响这些蛋白质与以下几种蛋白质的相互作用：(I)DNA、(Ii)衣壳蛋白、(Iii)切割/包装蛋白和(Iv)宿主细胞蛋白。该项目的具体目标是：1.确定UL25的衣壳位置、DNA结合特异性和蛋白质-蛋白质相互作用。2.通过对我们分离到的一系列HSV-1 UL28突变体的特征分析，确定UL28对于衣壳的整合、与病毒DNA包装位点的结合以及与终止酶(UL15)和门户蛋白(UL6)相互作用的重要功能结构域；并定位第二个位点突变，以使其中一个致命的UL28接头插入突变体回复，因为标记拯救和DNA测序表明该突变没有映射到UL28基因。3.利用蛋白质组学和生物化学方法研究UL25和UL28与病毒和细胞蛋白的相互作用，以确定参与切割/包装反应的基本蛋白质-蛋白质相互作用和蛋白质复合体。这些研究旨在阐明HSV DNA切割和包装的机制，并可能为抗病毒药物的开发提供新的靶点。

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.