How does a viral pathogen manipulate DNA Damage Responses to promote infection?

摘要 DNA病毒在受感染的细胞中诱导细胞DNA损伤反应(DDR)，无论是对 进入的病毒基因组，或在病毒复制过程中产生的大量外来基因组。这个 DDR形成了一种天生的关键屏障，可以阻碍或促进病毒复制。在宿主细胞感染后， 小鼠细小病毒微小病毒(MVM)的复制会诱导持续的DDR，然后被利用来 增强其可复制性。 细小病毒是一种无包膜的二十面体小病毒，是许多动物的重要病原体。 包括人类在内的物种。这些是唯一已知的脊椎动物病毒，含有单链线性 DNA基因组，在感染过程中向宿主细胞核呈现新的复制DNA结构，而 依靠细胞过程进行复制。Pintel实验室最近的研究表明，MVM与 DNA损伤的部位，以启动和放大其感染。此应用程序建议检查MVM如何 利用细胞DDR为有效接管细小病毒准备核环境。 目的1：MVM如何启动和激活宿主细胞的DNA损伤信号通路？MVM 感染在MRN(Mre11-Rad50-NBS1)全复合体中招募细胞DDR传感器Mre11- 独立方式，但激活下游ATM信令级联。本项目将研究如何 MRE11定位于MVM，并在没有其规范的中间适配器复合体的情况下启动下游DDR。 目标2：MVM如何利用DNA和蛋白质桥接分子建立复制中心 DNA损伤的部位？MVM与建筑蛋白CTCF结合的细胞DDR位点相关联，并且 DDR-染色质结合蛋白MDC1。本研究将破译CTCF和MDC1的作用机制 推动病毒复制中心的形成，以及它们如何影响细胞染色质结构。 目的3：确定MVM感染是如何引起宿主细胞DNA损伤的。MVM感染导致 细胞内产生的活性氧(ROS)，对细胞DNA的损伤起作用。这 研究将描述MVM如何诱导ROS，以及它如何利用产生的应激信号来放大 下游的DDR信号，从而扩大感染。 对传入的病毒基因组和细胞DDR传感器之间的相互作用的表征将提供 对蜂窝DDR级联如何启动的重要洞察。其次，它将阐明其作用机制。 MVM与宿主染色体在DNA损伤部位的反式作用。最后，关于ROS的研究 诱导将有助于理解病毒复制如何诱导额外的DNA断裂，从而使其 进一步扩大规模。研究这些过程对于了解细小病毒如何与 细胞，它们如何编程成功感染，以及最终它们如何导致疾病或作为基因疗法持续存在 车辆。这些研究的结果将可外推到其他DNA病毒和致病病毒系统。

SUMMARY DNA viruses elicit a cellular DNA damage response (DDR) in infected cells, either in response to incoming viral genomes, or to the large number of foreign genomes generated during virus replication. The DDR forms an innate, critical barrier that can impede or facilitate virus replication. Following host cell infection, replication of the parvovirus minute virus of mice (MVM) induces a sustained DDR, which is then exploited to enhance its replication. Parvovirinae, small non-enveloped icosahedral viruses, are important pathogens in many animal species including humans. These are the only known viruses of vertebrates that contain single-stranded linear DNA genomes, presenting novel replicative DNA structures to the host cell nucleus during infection while relying on cellular processes to replicate. Recent studies by the Pintel lab has shown that MVM interacts with sites of DNA damage to initiate and amplify its infection. This application proposes to examine how MVM exploits the cellular DDR to prepare the nuclear environment for effective parvovirus takeover. Aim 1: How does MVM initiate and activate the host cell’s DNA damage signaling pathway? MVM infection recruits the cellular DDR sensor MRE11 in an MRN (MRE11-RAD50-NBS1) holocomplex- independent manner, but activates a downstream ATM signaling cascade. This project will examine how MRE11 localizes to MVM, and initiates a downstream DDR without its canonical intermediate adaptor complex. Aim 2: How does MVM utilize DNA- and protein- bridging molecules to establish replication centers at sites of DNA damage? MVM associates with cellular DDR sites bound by the architectural protein CTCF, and the DDR-chromatin binding protein MDC1. This study will decipher the mechanism by which CTCF and MDC1 drive the formation of viral replication centers, and how they influence the cellular chromatin architecture. Aim 3: Determine how MVM infection induces additional host-cell DNA damage. MVM infection leads to the production of cellular reactive oxygen species (ROS), which contributes to cellular DNA damage. This research will characterize how MVM induces ROS, and how it utilizes the resulting stress signals to amplify downstream DDR signals, thereby expanding infection. Characterization of the interaction between the incoming viral genome and cellular DDR sensors will provide important insight into how cellular DDR cascades are initiated. Secondly, it will elucidate the mechanisms of trans-interaction between MVM and host chromosome at sites of DNA damage. Finally, studies on ROS induction will help in the understanding of how virus replication induces additional DNA breaks, allowing it to expand further. Examining these processes will be essential for understanding how parvoviruses interact with cells, how they program successful infection and ultimately how they cause disease or persist as gene therapy vehicles. Findings from these studies will be extrapolatable to other DNA viruses and pathogenic viral systems.