Viral Modulation of Genetic Stability

描述(申请人提供)：病毒为研究基本的细胞机制提供了强大的工具。以人腺病毒为模型，我们发现细胞DNA修复机制是病毒复制的障碍。细胞DNA修复途径在感染E4区缺失的病毒时产生双链病毒基因组的串联。Mre11/Rad50/NBS1复合体是串联体形成所必需的。这种细胞复合体对于双链DNA断裂修复、减数分裂重组和端粒维持都是重要的。Mre11和NBS1基因缺陷分别在AT样障碍(ATLD)和Nijmegen断裂综合征中易发生恶变。我们证明，腺病毒有两种机制来灭活这种细胞复合体。一种病毒蛋白(E4orf3)导致Mre11复合体的错误定位，另外两种病毒蛋白(E4orf6/E1b55K)导致Mre11和Rad50蛋白的降解。这些病毒蛋白通过“打了就跑”的策略防止了连锁反应，也导致了细胞转化。 我们建议调查Mre11复合体在串联形成中的作用，以及病毒用来靶向该复合体进行降解的机制。我们将使用生化和分子方法来确定病毒基因组串联所需的Mre11复合体的活性。我们将研究Mre11复合体与病毒蛋白的相互作用，并确定E1b55K/E4orf6复合体是否能泛化Mre11蛋白，导致其降解。我们观察到，感染缺乏E4orf6/E1b55K复合体的病毒会导致DNA修复信号通路的激活。我们将研究这些事件并确定它们是如何被E4orf6/E1b55K阻止的。我们还将研究Mre11降解对细胞的影响及其对DNA修复的影响。这些研究将为感知和响应DNA损伤的细胞途径提供洞察力，并将阐明病毒癌蛋白如何导致转化。

DESCRIPTION (provided by applicant): Viruses provide powerful tools to study fundamental cellular mechanisms. Using human adenovirus as a model we have identified the cellular DNA repair machinery as an obstacle to viral replication. Cellular DNA repair pathways produce concatemers of the double-stranded viral genome during infection with viruses deleted of the E4 region. The Mre11/Rad50/NBS1 complex is essential for concatemer formation. This cellular complex is important for double-strand DNA break repair, meiotic recombination and telomere maintenance. Defects in Mre11 and NBS1 genes predispose to malignancy in AT-like disorder (ATLD) and Nijmegen breakage syndrome respectively. We show that adenovirus possesses two mechanisms to inactivate this cellular complex. One viral protein (E4orf3) causes mislocalization of the Mre11 complex and two other viral proteins (E4orf6/E1b55K) induce degradation of Mre11 and Rad50 proteins. These viral proteins prevent concatemerization and also cause cell transformation using a "hit-and-run" strategy. We propose to investigate the role of the Mre11 complex in concatemer formation and the mechanism used by the virus to target this complex for degradation. We will use biochemical and molecular approaches to determine the activities of the Mre11 complex required for concatemerization of viral genomes. We will examine interactions of the Mre11 complex with the viral proteins and determine whether the E1b55K/E4orf6 complex can ubiquitinate the Mre11 protein to cause its degradation. We have observed that infection with viruses lacking the E4orf6/E1b55K complex results in activation of DNA repair signaling pathways. We will study these events and determine how they are blocked by E4orf6/E1b55K. We will also look at the consequences for the cell of Mre11 degradation and its effect on DNA repair. These studies will provide insights into cellular pathways that sense and respond to DNA damage and will also shed light on how viral oncoproteins cause transformation.