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-1)是一种重要的人类病原体，可导致免疫功能正常患者的自限性黏膜皮肤损害，以及新生儿和其他免疫功能低下者的潜在致命感染。在这项建议中，我们将测试假设，即单纯疱疹病毒利用重组依赖的复制，以复制其基因组使用病毒和细胞蛋白。我们认为，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，细胞通常使用这些机制来应对DNA损伤和其他应激因素。用来监测和修复受损DNA的细胞机制对维持基因组的稳定至关重要。暴露在DNA损伤剂下的哺乳动物细胞会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的影响。这些通路的缺陷会导致癌症等疾病。疱疹病毒与宿主共同进化，发展出引人入胜的避开、颠覆和在某些情况下受益于宿主细胞反应的方式。在这个提案中，我们将检验HSV利用同源重组(HR)修复途径为自己谋利的假设。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重组依赖的复制，这是产生后代基因组所必需的。我们先前已经证明，病毒的5‘到3’外切酶UL12和主要的单链DNA结合蛋白ICP8可以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重组酶的研究，并检验HSV通过病毒和细胞途径复制其基因组的假设。Aim 1将检验重组对产生病毒感染至关重要的假说；Aim 2将检验ICP8和UL12在感染过程中共同工作的假说；Aim 3将检验细胞重组蛋白被招募到病毒感染前部位和活跃复制中心并与其相互作用的假说。将结合使用遗传、生物物理、生化和细胞生物学方法。HSV-1是一种主要的人类病原体，对DNA复制的机制知之甚少。我们已经提出，该病毒利用了一些与细胞用于预防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是重要的，以便制定更好的治疗策略。在这项提议中，我们将检验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.