The parvovirus-induced DNA damage response and cell cycle perturbations

细小病毒诱导的 DNA 损伤反应和细胞周期扰动

• 批准号: 9185939
• 负责人: DAVID J. PINTEL
• 金额: $ 37.95万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9185939
• 关键词: Animals; Area; Biological Models; CDC2 Protein Kinase; Cell Communication; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell physiology; Cells; Cellular biology; Complex; DNA; DNA Damage; DNA Structure; DNA Viruses; DNA biosynthesis; DNA-Directed DNA Polymerase; Disabled Persons; Disease; Environment; G2 Phase; G2/M Arrest; Genome; Human; Income; Infection; Knowledge; Licensing Factor; Ligase; Mediation; Mediator of activation protein; Mice Minute Virus; Mitotic; Mus; Mutagens; Nuclear; Parvoviridae; Parvovirinae; Parvovirus; Play; Process; Proteins; Publishing; RNA; Replication Licensing; Replication-Associated Process; Role; Signal Transduction; Single-Stranded DNA; Source; Stimulus; System; TP53 gene; Time; Vertebrate Viruses; Viral; Viral Genome; Viral Nonstructural Proteins; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; Work; base; cell type; cyclin B1; design; gene therapy; insight; mouse genome; novel; pathogen; prevent; programs; public health relevance; repaired; response; ubiquitin-protein ligase

 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.

 描述（由申请人提供）：小鼠细小病毒微小病毒（MVM）的复制诱导持续的细胞DNA损伤反应（DDR），然后病毒利用该反应增强其在宿主细胞中的感染。MV诱导的DDR的一个重要方面是建立有效的G2/M期阻滞。虽然p53仍然被激活，但p21被降解，ATR/Chk 1信号被禁用。我们发现，一个令人惊讶的p21和Chk 1独立的细胞周期阻滞是通过一种新的机制，导致细胞周期蛋白B1及其编码RNA的显着，特异性的消耗，从而排除了细胞周期蛋白B1/CDK 1复合物的功能，从而防止有丝分裂进入。细小病毒亚科是小型无包膜二十面体病毒，是包括人类在内的许多动物物种的重要病原体。它们是脊椎动物中唯一已知的含有单链线性DNA基因组的病毒，并且它们在感染过程中不断向细胞呈现新的复制DNA结构。本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。以下目标是根据最近发表的研究结果确定的：MVM诱导的DDR如何以及出于何种目的导致CRL 4Cdt 2 E3泛素连接酶重新定位于APAR体？有效的MVM复制需要p21的CRL 4Cdt 2 E3泛素连接酶靶向消耗，以防止其与PCNA的抑制性相互作用，PCNA是复制MVM基因组的DNA聚合酶抑制剂的重要辅因子。这种连接酶还具有参与DNA复制的其他重要靶标。我们建议进一步表征病毒复制中心内的连接酶复合物的组分的状态和定位;表征p21被耗尽的机制，Cdt 1被保留，以及这如何促进感染;并调查感染期间连接酶的潜在额外作用。Aim II.如何以及出于什么目的，ATR信号在MVP诱导的DDR期间被禁用？Chk 1是ATR的下游靶点，通常是细胞周期停滞的重要介质，也不是ATR本身，在MVM感染期间被激活，即使携带结合RPA的病毒基因组-通常是ATR激活的有效触发剂-在病毒复制中心积累。现在清楚的是，ATR信令在MVP诱导的DDR建立之后被主动禁用。我们建议调查ATR被禁用的方法，以及在感染期间禁用ATR的目的。Aim III. MVM诱导的DDR是如何以及出于何种目的导致细胞周期蛋白B1的耗竭的？MVM诱导的p21和Chk 1非依赖性细胞周期停滞通过一个与其他DNA损伤剂或其他病毒感染不同的过程进行：它导致细胞周期蛋白B1及其编码RNA的急剧耗尽，从而阻止有丝分裂进入。我们建议确定的机制，其中细胞周期蛋白B1 RNA被耗尽在感染的细胞，是否有一个正在进行的DDR的背景下的病毒非结构蛋白的作用，并确定在感染过程中，这种消耗服务的目的。