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: 9028668
• 负责人: DAVID J. PINTEL
• 金额: $ 37.97万
• 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/9028668
• 关键词: Animals; Area; Binding; 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; Infection; Knowledge; Licensing Factor; Ligase; Mediation; Mediator of activation protein; Mice Minute Virus; Mitotic; Mus; Mutagens; Nuclear; Parvovirinae; Parvovirus; Parvovirus Infections; Play; Process; Proteins; Publishing; RNA; Replication Licensing; Research Design; 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; 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（通过应用提供）：小鼠（MVM）细小病毒分钟病毒的复制会诱导持续的细胞DNA损伤反应（DDR），然后该病毒利用该反应来增强其在宿主细胞中的感染。 MVM引起的DDR的一个重要方面是建立潜在的G2/m逮捕。尽管p53仍然激活，但p21被降解，并且禁用了ATR/CHK1信号。我们发现，通过一种新型机制建立了令人惊讶的P21-和CHK1独立的细胞周期块，该机制导致细胞周期蛋白B1的显着，特定的部署及其编码RNA，从而排除了Cyclin B1/CDK1复杂函数，从而防止了有丝分裂的进入。 Parvovirinae是小的非发育二十体病毒，在包括人类在内的许多动物物种中都是重要的病原体。它们是唯一含有单链线性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型的复制DNA结构。该应用建议进一步研究MVM如何探讨细胞DDR，以准备有效的细节性核环境。以下目的基于最近发表的发现：AIM I. MVM诱导的DDR如何以及出于什么目的会导致CRL4CDT2 E3泛素连接酶的重新定位到Apar身体？有效的MVM复制需要CRL4CDT2 E3泛素连接酶靶向P21的部署，以防止其与PCNA的抑制相互作用，PCNA是DNA聚合酶的必不可少的co contactor，这是复制MVM基因组的必不可少的。该连接酶还具有其他重要的DNA复制靶标。我们建议进一步表征病毒复制中心中连接酶复合物成分的状态和定位；表征p21耗尽的机制，cdt1被保存，以及如何促进感染。并研究感染过程中连接酶的潜在其他作用。目标II。在MVM诱导的DDR期间，ATR信号传导如何，出于什么目的？ CHK1是ATR的下游靶标，通常是细胞周期停滞的重要介质，也没有ATR本身在MVM感染过程中被激活，即使具有结合RPA的病毒基因组（通常是ATR激活的潜在触发） - 在病毒复制中心积聚。现在很明显，在建立MVM诱导的DDR后，ATR信号被积极禁用。我们建议研究ATR被禁用的手段，以及其在感染过程中残疾人的目的。目标三。 MVM诱导的DDR如何以及出于什么目的会导致细胞周期蛋白B1的耗竭？ MVM诱导的P21-和CHK1非依赖性细胞周期停滞通过一个过程与其他响应其他DNA受损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的急剧部署，从而阻止了有丝分裂的进入。我们建议确定细胞周期蛋白B1 RNA在感染细胞中耗尽的机制，是否在持续的DDR的背景下病毒非结构性蛋白具有作用，并确定该部署在感染过程中的目的。