Disabling of the Anaphase Promoting Complex by Human Cytomegalovirus

描述（由申请人提供）：人巨细胞病毒（HCMV）是导致出生缺陷的主要病毒，在免疫抑制个体中引起显著的发病率和死亡率，并被认为是血管疾病的一个危险因素。这种病毒已经进化出多种机制来篡夺细胞信号和调节途径，以促进其复制。一个关键的问题是，病毒如何激活细胞，使其达到最适合DNA复制的状态，同时改变选定的细胞蛋白质的水平和活性，从而以牺牲宿主为代价进行病毒复制。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。该病毒的一个特定靶标是多亚基后期促进复合体（APC） E3泛素连接酶。在感染的早期阶段，该复合体被禁用，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的过度磷酸化，APC1亚基和Cdh1与APC亚复合物核心（APC亚基3,6,7,8）的结合丧失以及APC亚基的重新定位。我们建议确定导致APC失调的病毒基因，并为此目的开发了一种高通量细胞测定方法。我们将采取两种互补的方法。第一个利用了与所有HCMV开放阅读框（orf）相对应的cDNA构建文库，第二个利用了在每个orf中都有突变的HCMV细菌人工染色体（BACs）文库。这些结果将为更深入地研究所涉及的分子和细胞机制以及这些基因在病毒复制和发病中的功能提供基础。公共卫生相关性：人类巨细胞病毒（HCMV）是导致出生缺陷的主要病毒，对免疫功能低下的个体构成严重问题，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控制其复制和与宿主相互作用的调控途径提供了重要动力。

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.