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-1）是一种重要的人类病原体，可在免疫功能正常的患者中引起自限性粘膜皮肤病变，并在新生儿和其他免疫功能低下的个体中引起潜在的致命感染。在本提案中，我们将验证HSV利用重组依赖复制利用病毒和细胞蛋白复制其基因组的假设。我们提出，HSV已经进化到与细胞修复和重组机制相互作用，细胞通常使用这些机制来应对DNA损伤和其他应激因素。用于监测和修复受损DNA的细胞机制对于维持基因组的稳定性至关重要。哺乳动物细胞暴露于DNA损伤剂诱导细胞周期检查点和DNA修复途径，以保护细胞免受突变和基因组重排。这些途径的缺陷会导致癌症等疾病。疱疹病毒与宿主共同进化，并发展出令人着迷的迂回方式，破坏宿主细胞的反应，有时甚至从宿主细胞的反应中获益。在本提案中，我们将验证HSV利用同源重组（HR）修复途径为其自身利益的假设。我们的初步工作表明，HSV使用病毒和细胞蛋白的组合来进行产生后代基因组所需的重组依赖复制。我们之前已经证明，病毒的5‘ to 3’核酸外切酶UL12和主要的单链DNA结合蛋白ICP8可以作为一个双亚基重组酶。我们建议继续我们对这种病毒重组酶的研究，并验证HSV病毒和细胞途径复制其基因组的假设。目的1将检验重组对多产性病毒感染至关重要的假设；目的2将验证ICP8和UL12在感染期间共同作用的假设；Aim 3将测试细胞重组蛋白被招募到病毒预增殖位点和活跃复制中心并与之相互作用的假设。将使用遗传、生物物理、生物化学和细胞生物学方法的结合。HSV-1是一种主要的人类病原体，对其DNA复制机制知之甚少。我们已经提出，病毒利用了一些细胞用来防止遗传不稳定和癌症的相同机制。由于DNA复制是抗病毒药物的主要靶点，因此充分了解这一过程中的关键因素对于制定更好的治疗策略非常重要。在这个提议中，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。

DESCRIPTION (provided by applicant): 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 DNA damage and other stress factors. The cellular machinery designed to monitor and repair damaged DNA is essential for maintaining genomic stability. Mammalian cells exposed to DNA damaging agents induce cell cycle checkpoints and DNA repair pathways that serve to protect the cell from mutations and genomic rearrangements. Defects in these pathways lead to diseases such as cancer. Herpesviruses have coevolved with 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 pathways for replication of its genome. Aim 1 will 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.