The parvovirus-induced DNA damage response and cell cycle perturbations

 描述(申请人提供)：复制小鼠细小病毒微小病毒(MVM)会诱导持续的细胞DNA损伤反应(DDR)，然后病毒利用这种反应来增强其在宿主细胞中的感染。MVM引发的解甲返乡的一个关键方面是建立强有力的G2/M逮捕。虽然p53仍然激活，但p21被降解，ATR/Chk1信号被禁用。我们发现，通过一种新的机制建立了令人惊讶的p21和Chk1独立的细胞周期阻滞，导致细胞周期蛋白B1及其编码的RNA显著、特异地耗尽，从而阻止了细胞周期蛋白B1/CDK1复合体的功能，从而阻止了有丝分裂的进入。细小病毒科是一种无包膜的二十面体小病毒，是包括人类在内的许多动物物种的重要病原体。它们是唯一已知的含有单链线性DNA基因组的脊椎动物病毒，并且在感染过程中不断向细胞呈现新的复制DNA结构。这项申请建议进一步研究MVM如何利用细胞DDR为有效的细小病毒接管准备核环境。以下目标基于最近发表的研究结果：目标I.MVM诱导的DDR如何以及出于什么目的导致CRL4CDt2 E3泛素连接酶重新定位到APAR小体？有效的巨噬细胞病毒复制需要CRL4Cdt2E3泛素连接酶靶向耗尽p21，以防止其与增殖细胞核抗原的抑制作用，增殖细胞核抗原是复制巨噬细胞病毒基因组的DNA聚合酶的重要辅助因子。这种连接酶还有其他与DNA复制有关的重要靶点。我们建议进一步表征连接酶复合体在病毒复制中心内的状态和定位；表征p21耗尽、CDT1幸免的机制，以及这如何促进感染；以及研究连接酶在感染过程中的潜在额外作用。目的II.在MVM诱导的DDR期间，ATR信号是如何被禁用的，目的是什么？Chk1是ATR的下游靶标，通常是细胞周期停滞的重要介质，在MVM感染期间，Chk1和ATR本身都不会被激活，尽管携带结合RPA的病毒基因组-通常是ATR激活的有效触发器-在病毒复制中心积累。现在清楚的是，在建立MVM诱导的DDR之后，ATR信号被主动禁用。我们建议调查ATR被禁用的方式，以及它在感染期间被禁用的目的。目的III.MVM诱导的DDR如何以及出于什么目的导致细胞周期蛋白B1的耗竭？MVM诱导的不依赖p21和Chk1的细胞周期停滞是通过一个不同于其他DNA损伤剂或其他病毒感染反应的过程进行的：它导致细胞周期蛋白B1及其编码的RNA急剧耗尽，从而阻止有丝分裂进入。我们建议确定周期蛋白B1 RNA在感染细胞中耗尽的机制，病毒非结构蛋白是否在正在进行的DDR中发挥作用，并确定这种耗尽在感染期间所起的作用。

 DESCRIPTION (provided by applicant): Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to enhance its infection in host cells. An essential aspect of the MVM-induced DDR is the establishment of a potent G2/M arrest. Although p53 remains activated, p21 is degraded and ATR/Chk1 signaling is disabled. We find that a surprising p21- and Chk1-independent cell cycle block is established via a novel mechanism that results in the significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function thus preventing mitotic entry. The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They are the only known viruses of vertebrates that contain single-stranded linear DNA genomes, and they continually present novel replicative DNA structures to cells during infection. This application proposes to further examine how MVM exploits the cellular DDR to prepare the nuclear environment for effective parvovirus takeover. The following aims are based on recently published findings: Aim I. How, and for what purpose, does the MVM-induced DDR cause re-localization of the CRL4Cdt2 E3 ubiquitin ligase to APAR bodies? Efficient MVM replication requires the CRL4Cdt2 E3 ubiquitin ligase-targeted depletion of p21 to prevent its inhibitory interaction with PCNA, an essential co-factor for DNA polymerase , which replicates the MVM genome. This ligase also has other important targets involved in DNA replication. We propose to further characterize the status and localization of the components of the ligase complex within viral replication centers; characterize the mechanism by which p21 is depleted, Cdt1 is spared, and how this facilitates infection; and investigate potential additional roles for the ligase during infection. Aim II. How, and for what purpose, is ATR signaling disabled during the MVM-induced DDR? Neither Chk1, a downstream target of ATR and normally an important mediator of cell cycle arrest, nor ATR itself, is activated during MVM infection, even though viral genomes bearing bound RPA - normally a potent trigger of ATR activation - accumulate in viral replication centers. It is now clear that ATR signaling is actively disabled following establishment of the MVM-induced DDR. We propose to investigate the means by which ATR is disabled, and the purpose its disabling serves during infection. Aim III. How, and for what purpose, does the MVM-induced DDR cause depletion of cyclin B1? The MVM-induced p21- and Chk1-independent cell cycle arrest proceeds via a process unlike that seen in response to other DNA-damaging agents or other virus infections: it results in a dramatic depletion of cyclin B1 and its encoding RNA which precludes mitotic entry. We propose to determine the mechanism by which cyclin B1 RNA is depleted in infected cells, whether there is a role for the viral nonstructural proteins within the context of an ongoing DDR, and to determine the purpose this depletion serves during infection.