Viral Modulation of Genetic Stability

项目摘要 病毒从它们感染的细胞中引发多方面的反应。在宿主的抗病毒防御中 细胞是一种DNA损伤反应的激活。在腺病毒(Ad)感染的情况下，双链、 线性DNA基因组代表细胞DNA修复途径的底物，导致Ad基因组 在感染E4开放阅读框缺失的病毒时加入串联蛋白。 连锁化需要宿主DNA修复蛋白，包括Mre11/Rad50/Nbs1复合体(MRN)。 感染E4缺失的Ad伴随着细胞DNA损伤信号级联的激活。 在野生型Ad感染过程中，DNA损伤反应被早期区域E4的产物灭活。 E4orf3蛋白在核内形成径迹结构，隔离MRN蛋白，防止ATR 损坏信号。E4orf6蛋白与E1b55K形成复合体，招募细胞蛋白形成 针对细胞修复因子进行降解的E3连接酶，包括MRN和连接酶IV蛋白。 本提案探索腺病毒感染后细胞反应的机制及其对 病毒生命周期。我们发现MRN抑制病毒DNA复制，确定CtIP蛋白的作用，以及 建议一种包括从病毒基因组中移除末端蛋白的模型。在第一个目标中，我们将 阐述细胞修复蛋白对病毒感染的影响，并测试该模型的抑制作用。这个 接下来的两个目标将解决E4蛋白中和这些宿主抗病毒功能的方式。在AIM 2我们将研究E1b55K/E4orf6靶向蛋白酶体降解的细胞因子的机制， 以及对病毒复制的影响。Aim 3将重点关注Ad E4orf3，并将调查两者之间的相关性 破坏PML小体，错位MRN复合体，防止串连形成，抑制 损伤信号和E4orf3产生晚期蛋白。这些研究将阐明 细胞修复因子与病毒蛋白相互作用的相关性，并将具有更广泛的 对我们理解细胞DNA损伤反应的影响。

Project Summary Viruses elicit multifaceted responses from the cells that they infect. Among the antiviral defenses of the host cell is activation of a DNA damage response. In the case of adenovirus (Ad) infection, the double-stranded, linear DNA genomes represent substrates for cellular DNA repair pathways that result in Ad genomes being joined into concatemers during infection with viruses deleted of the E4 open reading frames. Concatemerization requires host DNA repair proteins, including the Mre11/Rad50/Nbs1 complex (MRN). Infection with E4-deleted Ad is accompanied by activation of the cellular DNA damage signaling cascades. The DNA damage response is inactivated during wild-type Ad infection by the products of the early region E4. The E4orf3 protein forms intranuclear track structures, which sequester the MRN proteins and prevent ATR damage signaling. The E4orf6 protein forms a complex with E1b55K that recruits cellular proteins to form an E3 ligase that targets cellular repair factors for degradation, including MRN and ligase IV proteins. This proposal probes mechanistic aspects of the cellular response to adenovirus infection and its impact on the viral lifecycle. We show that MRN inhibits virus DNA replication, identify a role for the CtIP protein, and suggest a model that involves removal of the terminal protein from the virus genome. In the first Aim we will address the impact of the cellular repair proteins on virus infection and test the model for inhibition. The subsequent two Aims will address ways in which E4 proteins counteract these host antiviral functions. In Aim 2 we will study the mechanism used by E1b55K/E4orf6 to target cellular factors for proteasomal degradation, and the impact on virus replication. Aim 3 will focus on Ad E4orf3, and will investigate the correlation between disruption of PML bodies, mis-localization of the MRN complex, prevention of concatemer formation, inhibition of damage signaling, and production of late proteins by E4orf3. These studies will elucidate the biological relevance of the interactions between cellular repair factors and viral proteins, and will have broader implications for our understanding of cellular DNA damage responses.