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

基本信息

批准号：
7548622
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
项目类别：
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/7548622
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 DNA DNA Binding DNA Damage DNA Sequence Rearrangement DNA biosynthesis Defect Defective Viruses Development Disease 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 treatment strategy viral DNA

项目摘要

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）是一种重要的人类病原体，可导致皮肤粘膜自限性免疫功能正常患者的病变以及新生儿和其他患者的潜在致命感染免疫功能低下的个体。在本提案中，我们将测试 HSV 利用的假设使用病毒和细胞蛋白进行重组依赖性复制来复制其基因组。我们提出 HSV 已经进化到与细胞修复和重组机制相互作用。细胞通常用来对 DMA 损伤和其他应激因素做出反应。细胞机械旨在监测和修复受损的 DMA 对于维持基因组稳定性至关重要。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，从而保护 DNA 细胞免受突变和基因组重排的影响。这些途径的缺陷会导致疾病，例如癌症。疱疹病毒与宿主共同进化，并发展出令人着迷的回避方式，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 使用同源重组 (HR) 修复途径来获取自身利益。我们的初步研究表明，HSV 使用病毒和细胞蛋白的组合来执行产生后代基因组所需的重组依赖性复制。我们之前有过证明病毒 5' 至 3' 核酸外切酶 UL12 和主要单链 DNA 结合蛋白， ICP8，可以充当二亚基重组酶。我们建议继续对这种病毒进行研究重组酶并测试 HSV 病毒和细胞复制其基因组途径的假设。目标 1 将检验重组对于有效病毒感染至关重要的假设；目标 2 将测试假设 ICP8 和 UL12 在感染过程中协同工作；目标 3 将检验以下假设：细胞重组蛋白被招募到病毒前增殖位点并与病毒前增殖位点和活性位点相互作用。复制中心。遗传、生物物理、生物化学和细胞生物学方法的结合将被使用。 HSV-1 是一种主要的人类病原体，人们对 DNA 复制的机制知之甚少。我们有提出病毒利用了细胞用来防止遗传不稳定性的一些相同机制和癌症。由于 DNA 复制是抗病毒药物的主要靶标，因此充分了解 DNA 复制非常重要在此过程中的关键参与者，以便制定更好的治疗策略。在本提案中，我们将检验 HSV 使用病毒和细胞蛋白的组合来进行 DNA 复制的假设。