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) 是导致口腔、生殖器和威胁视力的眼部感染的主要病原体，对于免疫功能正常的成年人可能会危及生命，对于免疫系统受损的个体则更为严重。多联体 DNA 的生产是产生子代病毒的重要步骤，因为包装机器必须在衣壳化过程中识别长于单位长度的多联体；然而，人们对它们形成的机制知之甚少。尽管有人提出病毒基因组环化和滚环复制导致多联体的形成，但一些证据表明 HSV DNA 复制更为复杂，可能涉及重组依赖性复制，让人想起噬菌体 lambda 和 T4。我们之前已经证明 HSV 编码两个亚基病毒重组酶（UL12 和 ICP8）。此外，我们和其他人已经证明细胞 DNA 损伤反应 (DDR) 蛋白对传染性子代病毒的产生有积极和消极的影响。两个亚基病毒重组酶（UL12 和 ICP8）与多种 DDR 蛋白相互作用。现在已经清楚，DDR 蛋白至少在四种途径中发挥作用，其中三种需要一定程度的同源性（A-NHEJ、HR 和 SSA），另一种则不需要（C-NHEJ）。我们假设 HSV 在这种复杂的环境中导航，以确保产生可包装成传染性病毒的病毒基因组。中心假设是 HSV 感染需要激活一个或多个同源依赖性修复/重组途径，并且包括早期 E3 泛素连接酶 ICP0 和病毒重组酶（UL12 和 ICP8）在内的病毒蛋白会影响途径选择。此外，我们建议病毒蛋白使 C-NHEJ 途径失活，以促进末端切除，并且同源依赖性途径导致形成可加工成感染性病毒的多联体。在目标 1 中，我们将确定 HSV 感染激活和失活哪些细胞途径以及这些行为的后果。在目标 2 中，我们将测试 UL12 影响 DDR 信号传导并指导通路选择的假设。在目标 3 中，我们将检验以下假设：病毒 DNA 的结构取决于感染期间激活的修复途径，并且在缺乏 UL12 的情况下，不适当的途径选择会导致结构异常 DNA 的积累。预计这项研究将增进我们对病毒 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疗法。

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.