Role of viral and cellular recombination proteins in HSV DNA replication

描述(申请人提供)：单纯疱疹病毒(HSV)是口腔、生殖器和眼部感染的主要病原体，对具有免疫能力的成年人来说可能危及生命，在免疫系统受损的人中甚至更严重。连体DNA的产生是产生后代病毒的关键步骤，因为包装机械在包装过程中必须识别超过单位长度的连体；然而，对它们形成的机制了解很少。虽然有人提出了病毒基因组的环化和滚环复制导致连锁体的形成，但一些证据表明，HSV的DNA复制更复杂，可能涉及重组依赖的复制，这让人想起噬菌体lambda和T4。我们先前已经证明HSV编码一个两个亚单位的病毒重组酶(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既有积极的影响，也有消极的影响。两个亚单位病毒重组酶(UL12和ICP8)与多种DDR蛋白相互作用。现在已经清楚的是，DDR蛋白至少有四条途径发挥作用，其中三条需要一定的同源性(A-NHEJ、HR和SSA)，另一条不需要(C-NHEJ)。我们假设，HSV在这个复杂的环境中导航，以确保产生可以包装成传染性病毒的病毒基因组。核心假设是HSV感染需要激活一个或多个同源依赖的修复/重组途径，包括早期E3泛素连接酶ICP0和病毒重组酶(UL12和ICP8)在内的病毒蛋白影响途径选择。此外，我们认为C-NHEJ途径被病毒蛋白灭活，以促进末端切除和导致形成可加工成感染性病毒的连锁体的同源依赖途径。在目标1中，我们将确定哪些细胞通路被HSV感染激活和失活，以及这些作用的后果。在目标2中，我们将检验UL12影响DDR信号并指导通路选择的假设。在目标3中，我们将检验这一假设，即病毒DNA的结构取决于感染过程中激活的修复途径，并且在没有UL12的情况下，不适当的途径选择会导致结构异常的DNA积累。希望这项研究将加深我们对病毒DNA复制机制的理解，为基因组稳定和癌症生物学提供重要的细胞蛋白功能的信息，并有助于新的抗病毒疗法的开发。

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.