Disabling of the Anaphase Promoting Complex by Human Cytomegalovirus

人巨细胞病毒使后期促进复合物失活

• 批准号: 7712782
• 负责人: DEBORAH Hye SPECTOR
• 金额: $ 22.56万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-12 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7712782
• 关键词: Affect; Bacterial Artificial Chromosomes; Binding; Cell Communication; Cell Cycle; Cell Cycle Arrest; Cell Line; Cells; Cellular Assay; Complement; Complementary DNA; Complex; Congenital Abnormality; Cyclin-Dependent Kinases; Cytomegalovirus; Cytomegalovirus Infections; DNA biosynthesis; Degradation Pathway; Disabled Persons; Electroporation; Fibroblasts; Geminin; Gene Expression; Genes; Goals; Grant; Human; Immunocompromised Host; Individual; Infection; Laboratories; Libraries; Measures; Molecular; Molecular and Cellular Biology; Morbidity - disease rate; Mutation; Nuclear Protein; Nuclear Proteins; Open Reading Frames; Pathogenesis; Phase; Plaque Assay; Production; Proteins; Protocols documentation; Red nucleus structure; Regulatory Pathway; Rest; Risk Factors; Sampling; Signal Transduction; Time; Ubiquitin; Vascular Diseases; Viral; Viral Genes; Viral Pathogenesis; Viral Proteins; Virus; anaphase-promoting complex; base; cDNA Library; gene function; immunosuppressed; mortality; multicatalytic endopeptidase complex; mutant; protein function; public health relevance; reconstitution; research study; ubiquitin-protein ligase; viral DNA

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, causes significant morbidity and mortality in immunosuppressed individuals, and has been proposed as a risk factor in vascular disease. This virus has evolved multiple mechanisms to usurp cellular signaling and regulatory pathways to facilitate its replication. A key question has been how the virus activates the cell to a state that is optimal for DNA replication, and at the same time alters the levels and activity of selected cellular proteins so that viral replication proceeds at the expense of the host. We have found that one mechanism by which HCMV exploits the host cell involves the ubiquitin-proteasome degradation pathway. A specific target of the virus is the multisubunit Anaphase Promoting Complex (APC) E3 ubiquitin ligase. During the early phase of the infection, this complex is disabled, allowing stabilization and accumulation of its substrates. The effects on the APC include degradation of the APC4 and APC5 subunits, hyperphosphorylation of Cdh1, a loss of binding of both the APC1 subunit and Cdh1 to subcomplex core of the APC (APC subunits 3, 6, 7, 8) and relocalization of the APC subunits. We propose to determine the viral gene(s) that are responsible for this dysregulation of the APC and have developed a high throughput cellular assay for this purpose. There are two complementary approaches that we will take. The first utilizes a library of cDNA constructs corresponding to all of the HCMV open reading frames (ORFs), and the second utilizes a library of HCMV Bacterial Artificial Chromosomes (BACs) that have mutations in each of the ORFs. These results will provide the basis for more in-depth studies on the molecular and cellular mechanisms involved and the functions of these genes with respect to viral replication and pathogenesis. PUBLIC HEALTH RELEVANCE: Human Cytomegalovirus (HCMV) is the major viral cause of birth defects, poses a serious problem for immunocompromised individuals, and has been proposed to be a risk factor for vascular disease. The serious problems associated with HCMV infections have provided a major impetus for understanding the molecular and cellular biology of the virus and the regulatory pathways governing its replication and interactions with the host.

描述（由申请人提供）：人类巨细胞病毒（HCMV）是先天性缺陷的主要病毒原因，在免疫抑制个体中引起明显的发病率和死亡率，并已被认为是血管疾病的危险因素。该病毒已将多种机制发展为篡夺细胞信号传导和调节途径，以促进其复制。一个关键问题是该病毒如何将细胞激活到最佳的DNA复制状态，同时改变选定的细胞蛋白的水平和活性，从而以宿主为代价进行病毒复制进行。我们发现，HCMV利用宿主细胞的一种机制涉及泛素 - 蛋白酶体降解途径。病毒的特定靶标是促进复合物（APC）E3泛素连接酶。在感染的早期阶段，该复合物被禁用，允许其底物的稳定和积累。对APC的影响包括APC4和APC5亚基的降解，CDH1的高磷酸化，APC1亚基和CDH1与APC的子复合核的结合丧失（APC亚基3、6、7、8）以及APC亚基的重新定位。我们建议确定导致APC失调的病毒基因，并为此而产生高吞吐量细胞测定法。我们将采用两种互补的方法。第一个利用与所有HCMV开放式阅读框（ORF）相对应的cDNA构建体库，第二个使用HCMV细菌人工染色体（BAC）的库，这些库中每个ORF中都有突变。这些结果将为对涉及的分子和细胞机制以及这些基因在病毒复制和发病机理方面的功能进行更深入的研究提供基础。公共卫生相关性：人类巨细胞病毒（HCMV）是出生缺陷的主要病毒原因，对免疫功能低下的个体构成了一个严重的问题，并被认为是血管疾病的危险因素。与HCMV感染相关的严重问题为理解病毒的分子和细胞生物学以及管理其复制和与宿主相互作用的调节途径提供了主要动力。