Role of viral and cellular recombination proteins in HSV DNA replication

描述（由申请人提供）：单纯疱疹病毒（HSV）是口腔、生殖器和威胁视力的眼部感染的主要病原体，在免疫功能正常的成年人中可危及生命，在免疫系统受损的个体中甚至更严重。串联DNA的产生是子代病毒产生的必要步骤，因为包装机械在封装过程中必须识别超过单位长度的串联体；然而，人们对它们形成的机制知之甚少。虽然已经提出病毒基因组环状和滚环复制导致串联体的形成，但一些证据表明HSV DNA复制更复杂，可能涉及重组依赖复制，使人想起噬菌体lambda和T4。我们之前已经证明HSV编码一个双亚基病毒重组酶（UL12和ICP8）。此外，我们和其他人已经证明细胞DNA损伤反应（DDR）蛋白对感染性子代病毒的产生既有积极的影响，也有消极的影响。两个亚基病毒重组酶（UL12和ICP8）与几种DDR蛋白相互作用。现在很清楚，DDR蛋白至少在四种途径中起作用，其中三种需要一定程度的同源性（A-NHEJ， HR和SSA），另一种不需要（C-NHEJ）。我们假设HSV导航这种复杂的环境，以确保产生可以包装成感染性病毒的病毒基因组。核心假设是HSV感染需要激活一种或多种同源依赖性修复/重组途径，病毒蛋白包括即时早期E3泛素连接酶ICP0和病毒重组酶（UL12和ICP8）影响途径选择。此外，我们认为C-NHEJ通路被病毒蛋白灭活，以促进末端切除和同源依赖通路的形成，从而形成可加工成感染性病毒的串联体。在目标1中，我们将确定哪些细胞途径被HSV感染激活和灭活，以及这些行为的后果。在Aim 2中，我们将检验UL12影响DDR信号传导并指导通路选择的假设。在目标3中，我们将验证病毒DNA的结构取决于感染期间激活的修复途径的假设，并且在缺乏UL12的情况下，不适当的途径选择导致结构异常DNA的积累。期望这项研究能够增进我们对病毒DNA复制机制的理解，提供对基因组稳定性和癌症生物学重要的细胞蛋白功能的信息，并有助于开发新的抗病毒治疗方法。

DESCRIPTION (provided by applicant): Herpes Simplex Virus (HSV) is a major pathogen responsible for oral, genital and sight-threatening ocular infections which can be life threatening in immunocompetent adults and even more severe in individuals with compromised immune systems. Production of concatemeric DNA is an essential step for the generation of progeny virus as the packaging machinery must recognize longer-than-unit-length concatemers during encapsidation; however, the mechanism by which they are formed is very poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication is more complex and may involve recombination-dependent replication reminiscent of bacteriophages lambda and T4. We have previously shown that HSV encodes a two-subunit viral recombinase (UL12 and ICP8). Furthermore cellular DNA Damage Response (DDR) proteins have been shown by us and others to both positively and negatively influence the production of infectious progeny virus. The two subunit viral recombinase (UL12 and ICP8) interacts with several DDR proteins. It is now clear that DDR proteins function in at least four pathways, three that require some amount of homology (A-NHEJ, HR and SSA) and one that does not (C-NHEJ). We hypothesize that HSV navigates this complex environment to ensure the production of viral genomes that can be packaged into infectious virus. The central hypothesis is that HSV infection requires the activation of one or more of the homology dependent repair/recombination pathways and that viral proteins including the immediate early E3 ubiquitin ligase ICP0 and the viral recombinase (UL12 and ICP8) act to influence pathway choice. Furthermore we suggest that the C-NHEJ pathway is inactivated by viral proteins in order to promote end resection and the homology dependent pathways leading to the formation of concatemers that can be processed into infectious virus. In aim 1, we will determine which cellular pathways are activated and inactivated by HSV infection and the consequences of these actions. In Aim 2 we will test the hypothesis that UL12 affects DDR signaling and directs pathway choice. In aim 3 we will test the hypothesis that the structure of viral DNA depends on the repair pathway activated during infection and that in the absence of UL12, inappropriate pathway choice leads to the accumulation of structurally aberrant DNA. It is expected that this study will improve our understanding of the mechanism of viral DNA replication, provide information on the functions of cellular proteins important for genome stability and cancer biology and aid in the development of new antiviral therapies.