FUNCTION OF HSP70 HOMOLOG ENCODED IN RNA VIRUS

RNA病毒中编码的HSP70同源物的功能

• 批准号: 6180930
• 负责人: VALERIAN V. DOLJA
• 金额: $ 18.89万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180930
• 关键词: RNA virus; adenosinetriphosphatase; affinity chromatography; enzyme activity; gene expression; gene mutation; immunocytochemistry; plant virus; protein localization; protein structure function; recombinant virus; site directed mutagenesis; stress proteins; transfection; virus infection mechanism; virus protein; yeast two hybrid system

DESCRIPTION: The major objective of this project is to elucidate the functions of heat shock proteins from the HSP70 family in viral pathogenesis. The cellular HSP70s are vital in stress responses, protein folding, translocation, and assembly, acting via protein-protein interactions modulated by an ATPase activity. Divers RNA and DNA viruses, including human pathogens, recruit HSP70s at various stages of their life cycle, or induce HSP70s following viral infection. However, the general role of protein chaperones in virus-host interactions is unknown. Beet yellows closterovirus (BYV) employed in this project encodes a HSP70-homolog that is essential for virus translocation from cell to cell. Use of BYV as a model provides unprecedented opportunity to directly probe the functions and mechanisms of HSP70h action in viral pathogenesis. Three specific aims of this project will address structure/function relations, subcellular distribution, and regulation of HSP70h expression. A. Functional dissection of HSP70h. Functionally important structural elements, role of the ATPase activity, and specialization of the HSP70h domains will be characterized using reverse genetic and biochemical approaches. Structural conservation among cellular and viral HSP70s will be used to design mutations that will be introduced into the full-length BYV cDNA. The availability of BYV tagged by GUS or GFP reporters will facilitate functional study of mutant phenotypes. B. Protein partners of HSP70h. Since all cellular HSP70s operate via protein-protein interactions, identification of target protein of HSP70h will provide important clues as to the mechanism of HSP70h action. Partner proteins of HSP70h will be identified using affinity chromatography, immunohistochemistry, and in vivo assays provided by the yeast two-hybrid system. C. Subcellular distribution and regulation of HSP70h. Localization of the HSP70h in infected cells and tissues will be examined by an immunohistochemical approach. In addition, HSP70h-GFP fusions produced by modified viruses will be utilized to reveal HSP70h distribution in live cells. The transcriptional and translational regulation of HSP70h will be studied in protoplasts transfection experiments using recombinant BYV expressing a reporter gene under control of the HSP70h promoter.

描述：该项目的主要目标是阐明 HSP70 家族热休克蛋白在病毒中的功能 发病。 细胞 HSP70 在应激反应、蛋白质 折叠、易位和组装，通过蛋白质-蛋白质起作用 由 ATP 酶活性调节的相互作用。 多种RNA和DNA病毒， 包括人类病原体，在其生命的各个阶段招募 HSP70 循环，或在病毒感染后诱导 HSP70。 然而，一般 蛋白质伴侣在病毒-宿主相互作用中的作用尚不清楚。 甜菜 该项目中使用的黄线状病毒 (BYV) 编码 HSP70 同源物 这对于病毒从一个细胞转移到另一个细胞至关重要。 使用 BYV 作为 模型提供了前所未有的机会来直接探索功能 以及 HSP70h 在病毒发病机制中的作用机制。 三个具体目标 该项目将解决结构/功能关系、亚细胞 HSP70h 表达的分布和调节。 A. 功能剖析 HSP70h。 功能上重要的结构元件，ATP 酶的作用 HSP70h 结构域的活性和专业化将得到表征 使用反向遗传和生化方法。 结构保护 细胞和病毒 HSP70 之间的突变将被用来设计突变 被引入全长BYV cDNA。 BYV 标记的可用性 GUS 或 GFP 报告基因将促进突变体的功能研究 表型。 B. HSP70h 的蛋白质伴侣。 由于所有蜂窝 HSP70 通过蛋白质-蛋白质相互作用进行操作，识别目标蛋白质 HSP70h 的表达将为了解 HSP70h 的作用机制提供重要线索。 HSP70h 的伴侣蛋白将使用亲和层析进行鉴定， 免疫组织化学和酵母双杂交提供的体内测定 系统。 C. HSP70h 的亚细胞分布和调节。 本土化 受感染细胞和组织中的 HSP70h 将由 免疫组织化学方法。 此外，HSP70h-GFP 融合体由 修改后的病毒将用于揭示 HSP70h 在活体中的分布 细胞。 HSP70h 的转录和翻译调控将是 使用重组 BYV 进行原生质体转染实验研究 在 HSP70h 启动子的控制下表达报告基因。