Modulation of herpes simplex virus type 1 genome structure during lytic replication

单纯疱疹病毒1型(HSV-1)是一种流行的病原体，感染大多数人类人群。病毒生命周期的大部分发生在受感染细胞的细胞核内，病毒基因组上发生的事件决定了感染的结果。然而，病毒DNA的结构如何对关键的病毒过程起到调节作用还不是很清楚。病毒粒子HSV-1 DNA以单链断裂和单链间隙进入宿主细胞。在病毒DNA复制过程中，分支基因组结构形成，很可能是重组介导的复制中间产物。我们以前曾证明，在感染早期和病毒DNA复制开始后，不同的细胞DNA修复蛋白组与病毒基因组相关。DNA损伤是如何通过病毒DNA复制、转录和包装机械来引导的，目前还不清楚。此外，HSV-1基因组的全基因组结构在感染的时间进程中如何被修改还没有定义。以前研究这些问题的一个主要障碍是无法探测全基因组DNA结构。在这个探索性的提案中，我们描述了我们的计划，即开发在整个生产性感染过程中定义HSV-1基因组结构的方法。这项提案中概述的目标将建立新的方法来绘制HSV-1DNA上的单链和双链断裂图，并研究病毒在体内的复制和重组中间产物。这些结果将为HSV-1转录、DNA复制、重组和修复的调控机制提供更深入的了解。此外，所生成的工具可用于探测HSV-1重新激活前后的基因组、缺乏特定细胞DNA修复和DNA损伤反应因子的细胞中的病毒感染以及其他DNA病毒的基因组。

Herpes simplex virus type 1 (HSV-1) is a prevalent pathogen that infects the majority of the human population. Much of the virus life cycle occurs in the nucleus of infected cells, where events that occur on the viral genome determine the outcome of infection. However, how the structure of the viral DNA contributes to the regulation of key viral processes is not well understood. Virion HSV-1 DNA enters into host cells containing single strand breaks and single stranded gaps. During viral DNA replication, branched genome structures form, likely as recombination-mediated replication intermediates. We previously demonstrated that distinct groups of cellular DNA repair proteins associate with viral genomes early during infection and after the onset of viral DNA replication. How DNA damage is navigated by viral DNA replication, transcription, and packaging machinery is not understood. Furthermore, how the genome-wide structure of the HSV-1 genome is modified during the temporal progression of infection has not been defined. A major roadblock to previously investigate these questions was the inability to probe genome-wide DNA structure. In this exploratory proposal, we describe our plan to develop approaches to define the structure of the HSV-1 genome throughout productive infection. The aims outlined in this proposal will establish new approaches to map single and double strand breaks on HSV-1 DNA and investigate viral replication and recombination intermediates in vivo. Results will provide greater insight into mechanisms of regulation of HSV-1 transcription, DNA replication, recombination, and repair. Furthermore, tools generated could be adapted to probe HSV-1 genomes before and after reactivation, viral infection in cells deficient for select cellular DNA repair and DNA damage response factors, and genomes of other DNA viruses.