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: 7338346
• 负责人: SANDRA K WELLER
• 金额: $ 28.2万
• 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 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7338346
• 关键词: Active Sites; Address; Affect; Antiviral Agents; Antiviral Therapy; Biochemical; Biological; Biological Assay; Cancer Biology; Cell Cycle Checkpoint; Cell Line; Cell physiology; Cells; Complex; Condition; DNA; DNA Binding; DNA Damage; DNA Sequence Rearrangement; DNA biosynthesis; DNA chemical synthesis; Defect; Defective Viruses; Development; Disease; Endopeptidases; Filament; Fluorescence; Frequencies; Genetic; Genetic Recombination; Genome; Genome Stability; Genomics; Goals; Growth; Herpesviridae; Herpesvirus 1; Human; Immunocompetent; Immunocompromised Host; In Vitro; Individual; Infection; Knock-out; Lead; Lesion; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Methods; Monitor; Mutation; Pathway interactions; Patients; Peptide Hydrolases; Plasmids; Process; Proteins; Proteolysis; Pulsed-Field Gel Electrophoresis; Recruitment Activity; Resistance; Roentgen Rays; Role; SS DNA BP; Side; Simplexvirus; Site; Small Interfering RNA; Spectrum Analysis; Stress; Structure; Testing; Transfection; Viral; Viral Proteins; Virus; Virus Diseases; Work; analytical ultracentrifugation; base; design; fascinate; gene replacement; homologous recombination; improved; in vivo; mutant; neonate; pathogen; prevent; recombinase; recombinational repair; repaired; response; spleen exonuclease; stoichiometry

Herpes simplex virus (HSV-1) is an important human pathogen responsible for self-limiting mucocutaneous lesions in immunocompetent patients and potentially lethal infections in neonates and other immunocompromised individuals. In this proposal we will test the hypothesis that HSV utilizes recombination-dependent replication to replicate its genome using both viral and cellular proteins. We propose that HSV has evolved to interact with the cellular repair and recombination machinery which the cell normally uses to respond to DMAdamage and other stress factors. The cellular machinery designed to monitor and repair damaged DMA is essential for maintaining genomic stability. Mammalian cells exposed to DNA damaging agents induce cell cycle checkpoints and DMA repair pathwaysthat serve to protect the cell from mutations and genomic rearrangements. Defects in these pathways lead to diseases such as cancer. Herpesviruses have coevolvedwith their hosts and developed fascinating ways of side stepping, subverting and in some cases benefiting from host cell responses. In this proposal we will test the hypothesis that HSV uses the homologous recombination (HR) repair pathway for its own benefit. Our preliminary work suggests that HSV uses a combination of viral and cellular proteins to carry out recombination-dependent replication needed to generate progeny genomes. We have previously demonstrated that the viral 5' to 3' exonuclease, UL12 and the major single strand DNA binding protein, ICP8, can function as a two-subunit recombinase. We propose to continue our studies of this viral recombinase and to test the hypothesis that HSV viral and cellular pathwaysfor replication of its genome. Aim 1will test the hypothesis that recombination is essential for productive viral infection; Aim 2 will test the hypothesis that ICP8 and UL12 work together during infection; and Aim 3 will test the hypothesis that cellular recombination proteins are recruited to and interact with viral preprelicative sites and active replication centers. A combination of genetic, biophysical, biochemical and cell biological approaches will be used. HSV-1 is a major human pathogen, and little is known about the mechanism of DNA replication. We have proposed that the virus utilizes some of the same machinery that cells use for preventing genetic instability and cancer. Since DNA replication is a major target for antiviral drugs, it is important to fully understand the key players in this process in order to develop better strategies for treatment. In this proposal we will test the hypothesis that HSV uses a combination of viral and cellular proteins to carry out DNA replication.

单纯疱疹病毒(HSV-1)是引起自限性皮肤粘膜的重要病原体 免疫活性患者的损害和新生儿和其他人的潜在致命性感染 免疫功能受损的个体。在这个提案中，我们将检验HSV利用 依赖重组的复制，使用病毒和细胞蛋白复制其基因组。我们 提出单纯疱疹病毒已经进化为与细胞修复和重组机制相互作用 细胞通常用来对DMAdamage和其他压力因素做出反应。蜂窝机械设计用于 监测和修复受损的DNA是维持基因组稳定所必需的。暴露的哺乳动物细胞 DNA损伤剂诱导细胞周期检查点和DMA修复途径，以保护 来自突变和基因组重排的细胞。这些通路的缺陷会导致疾病，如 癌症。疱疹病毒与宿主共同进化，形成了引人入胜的躲避方式， 颠覆宿主细胞反应，并在某些情况下受益于宿主细胞反应。在本提案中，我们将测试 假设HSV利用同源重组(HR)修复途径为自己谋利。我们的 初步研究表明，单纯疱疹病毒使用病毒和细胞蛋白的组合来进行 重组依赖的复制需要产生后代基因组。我们之前已经 证明了病毒的5‘至3’外切酶、UL12和主要单链DNA结合蛋白， ICP8可作为双亚基重组酶发挥作用。我们建议继续我们对这种病毒的研究 重组酶，并验证HSV病毒和细胞途径复制其基因组的假设。 目标1将检验重组对生产性病毒感染至关重要的假设；目标2将检验 假设ICP8和UL12在感染期间一起工作；目标3将测试该假设 细胞重组蛋白被招募到病毒先发部位并与之相互作用，并具有活性 复制中心。遗传、生物物理、生化和细胞生物学方法的组合将 被利用。 HSV-1是一种主要的人类病原体，对DNA复制的机制知之甚少。我们有 提出该病毒利用了一些与细胞用来防止遗传不稳定相同的机制 和癌症。由于DNA复制是抗病毒药物的主要靶点，因此充分了解 在这一进程中的关键参与者，以便制定更好的治疗策略。在本提案中，我们将 测试HSV使用病毒和细胞蛋白质的组合来进行DNA复制的假设。