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（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来增强其在宿主细胞中的感染，这是MVM诱导的DDR的重要方面。是建立有效的 G2/M 停滞，尽管 p53 保持激活，但 p21 被降解并且 ATR/Chk1 信号传导被禁用，我们发现一个令人惊讶的 p21 和 Chk1 独立细胞。循环阻断是通过一种新机制建立的，该机制导致细胞周期蛋白 B1 及其编码 RNA 的显着、特异性消耗，从而阻止细胞周期蛋白 B1/CDK1 复合体功能，从而阻止有丝分裂进入。细小病毒亚科是小型无包膜二十面体病毒，是重要的病原体。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，并且不断地呈现新的复制 DNA。该申请建议进一步研究 MVM 如何利用细胞 DDR 来准备细胞核环境，以实现有效的细小病毒接管。 MVM 诱导的 DDR 导致 CRL4Cdt2 E3 泛素连接酶重新定位到 APAR 体？有效的 MVM 复制需要 CRL4Cdt2 E3 泛素吗？连接酶靶向消除 p21，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  的重要辅助因子，该连接酶还具有参与 DNA 复制的其他重要靶标。连接酶复合物成分在病毒复制中心内的定位；描述 p21 被耗尽、Cdt1 免受影响的机制，以及这如何促进感染；目的 II. 在 MVM 诱导的 DDR 过程中，ATR 信号传导是如何以及出于什么目的而被禁用的？ MVM 感染，尽管带有结合 RPA（通常是 ATR 激活的有效触发因素）的病毒基因组在病毒复制中心积聚，但现在很清楚，在 MVM 诱导的 DDR 建立后，ATR 信号传导被主动禁用。目的 III. MVM 诱导的 DDR 是如何导致细胞周期蛋白 B1 耗竭的？其进展过程与其他 DNA 损伤剂或其他病毒感染的反应过程不同：它导致细胞周期蛋白 B1 及其编码 RNA 的急剧消耗，从而阻止有丝分裂进入。我们建议确定感染细胞中细胞周期蛋白 B1 RNA 消耗的机制，病毒非结构蛋白在持续 DDR 的背景下是否发挥作用，并确定这种消耗在感染期间的目的。