Role of cdks and dynein in HCMV Assembly

cdks 和动力蛋白在 HCMV 组装中的作用

• 批准号: 6672681
• 负责人: VERONICA SANCHEZ
• 金额: $ 10.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-18 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6672681
• 关键词: cyclin dependent kinase; cytomegalovirus; dynein ATPase; electron microscopy; enzyme activity; immunofluorescence technique; immunoprecipitation; intracellular transport; microtubule associated protein; microtubules; protein localization; protein structure function; virus assembly

DESCRIPTION (provided by applicant): The goal of this application is to provide continuing support for Dr. Veronica Sanchez' training as a research scientist in the field of herpesvirology. Phase I of the proposed work will cultivate her research and teaching skills in a mentored environment in the laboratory of Dr. Deborah Spector at UCSD. Phase II will mark her transition to an independent research position in academia. Microtubule-based transport plays an important role in many cellular processes. Cytoplasmic dynein is a multimeric protein that, in association with the dynactin complex, mediates the transport of membranous and non-membranous cargo toward the microtubule-organizing center (MTOC) at the centrosome. Because the site of human cytomegalovirus (HCMV) envelopment overlaps the MTOC, we propose that dynein is involved in the transport of nucleocapsids from the nucleus to the cytoplasmic assembly compartment. To address this hypothesis, the expression and localization of dynein and dynactin subunits in HCMV-infected fibroblasts will be established. In addition, a functional analysis of dynein activity will be undertaken. Dynein activity will be inhibited in HCMV-infected cells by over-expression of dynactin subunits or small interfering RNAs (siRNAs) from recombinant viruses. The effects of dynein inhibition on the transport of nucleocapsids to the cytoplasmic assembly compartment will be determined by electron microscopy and immunofluorescence assays. The regulation of dynein activity by the cyclin-dependent kinases (cdks) in infected cells will also be studied. Recombinant HCMV viruses expressing dominant-negative forms of the cdks or siRNAs targeting the cyclins will be used to study the effect of phosphorylation on dynein activity in infected cells. These studies should provide valuable information regarding the role of dynein and cdk activity in HCMV assembly. In addition, the results from these studies will provide the basis for Dr. Sanchez to develop an independent research program and assist her to secure a faculty position at a university or medical school.

描述(由申请人提供)：本申请的目标是为Veronica Sanchez博士作为疱疹病毒学领域的研究科学家的培训提供持续支持。拟议工作的第一阶段将在加州大学圣地亚哥分校黛博拉·斯佩克特博士的实验室指导环境中培养她的研究和教学技能。第二阶段将标志着她过渡到学术界的独立研究职位。 基于微管的转运在许多细胞过程中发挥着重要作用。细胞质动力蛋白是一种多聚体蛋白，与动力蛋白复合体结合，介导膜性和非膜性物质向中心体的微管组织中心(MTOC)的运输。由于人巨细胞病毒(HCMV)被膜部位与MTOC重叠，我们推测动力蛋白参与了核衣壳从细胞核到细胞质组装间的运输。为了解决这一假设，将建立动力蛋白和动力蛋白亚基在人巨细胞病毒感染的成纤维细胞中的表达和定位。此外，还将进行动力蛋白活性的功能分析。在HCMV感染的细胞中，动力蛋白亚基或重组病毒的小干扰RNA(SiRNAs)的过度表达将抑制动力蛋白的活性。动力蛋白抑制对核衣壳转运到细胞质集合室的影响将通过电子显微镜和免疫荧光分析来确定。还将研究感染细胞中细胞周期蛋白依赖性蛋白激酶(CDKs)对动力蛋白活性的调节。表达针对细胞周期蛋白的显性-阴性形式的CDKs或siRNAs的重组HCMV病毒将被用来研究磷酸化对感染细胞中动力蛋白活性的影响。这些研究应该提供关于动力蛋白和CDK活性在HCMV组装中的作用的有价值的信息。此外，这些研究的结果将为桑切斯博士开发独立的研究项目提供基础，并帮助她在大学或医学院获得教职。