Role of viral and cellular recombination proteins in HSV DNA replication

病毒和细胞重组蛋白在 HSV DNA 复制中的作用

• 批准号: 8438424
• 负责人: SANDRA K WELLER
• 金额: $ 32.21万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8438424
• 关键词: Adult; Affect; Antiviral Therapy; Bacteriophage lambda; Biochemical; Biological Assay; Cancer Biology; Cell physiology; Cells; Complex; DNA; DNA Damage; DNA biosynthesis; Development; Double Strand Break Repair; Ensure; Environment; Excision; Eye Infections; G22P1 gene; Generations; Genetic Recombination; Genital system; Genome Stability; Goals; Growth; Head; Herpesvirus 1; Human; Human Herpesvirus 2; Immune system; Immunocompetent; In Vitro; Individual; Infection; Lead; Length; Life; Maintenance; Measures; Mediating; Morbidity - disease rate; Mutation; Nonhomologous DNA End Joining; Oral; Pathway interactions; Plasmids; Process; Production; Proteins; Recombinants; Recruitment Activity; Reporter; Resolution; Role; Signal Transduction; Simplexvirus; Structure; Surgical Flaps; Tail; Testing; Transfection; Viral; Viral Genes; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; Vision; base; improved; irradiation; mortality; mutant; novel; nuclease; pathogen; protein function; recombinase; recombinational repair; repaired; response; ubiquitin-protein ligase; viral DNA

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.

描述（由申请方提供）：单纯疱疹病毒（HSV）是一种主要病原体，可引起口腔、生殖器和危及视力的眼部感染，在免疫功能正常的成人中可能危及生命，在免疫系统受损的个体中甚至更严重。多联体DNA的产生是产生子代病毒的重要步骤，因为包装机器必须在双核苷化过程中识别比单位长度长的多联体;然而，对它们形成的机制知之甚少。虽然已经提出病毒基因组环化和滚环复制导致多联体的形成，但几条证据表明HSV DNA复制更复杂，可能涉及重组依赖性复制，使人联想到λ和T4噬菌体。我们先前已经证明HSV编码两个亚基病毒重组酶（UL 12和ICP 8）。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有正面和负面影响。两个亚基病毒重组酶（UL 12和ICP 8）与几种DDR蛋白相互作用。现在清楚的是，DDR蛋白在至少四种途径中起作用，三种需要一定量的同源性（A-NHEJ、HR和SSA），一种不需要同源性（C-NHEJ）。我们假设HSV在这种复杂的环境中导航，以确保病毒基因组的产生，这些基因组可以被包装成感染性病毒。中心假设是HSV感染需要激活一种或多种同源依赖性修复/重组途径，并且包括立即早期E3泛素连接酶ICP 0和病毒重组酶（UL 12和ICP 8）的病毒蛋白质起作用以影响途径选择。此外，我们认为，C-NHEJ途径被病毒蛋白失活，以促进末端切除和同源依赖性途径，导致可以加工成感染性病毒的多联体的形成。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。在目标2中，我们将测试UL 12影响DDR信号传导并指导途径选择的假设。在目标3中，我们将测试病毒DNA的结构取决于感染期间激活的修复途径的假设，并且在不存在UL 12的情况下，不适当的途径选择导致结构异常DNA的积累。预计这项研究将提高我们对病毒DNA复制机制的理解，提供有关对基因组稳定性和癌症生物学重要的细胞蛋白质功能的信息，并有助于开发新的抗病毒疗法。