FUNCTION OF HSP70 HOMOLOG ENCODED IN RNA VIRUS

描述：本项目的主要目标是阐明 HSP 70家族热休克蛋白在病毒感染中的作用 发病机制 细胞HSP 70在应激反应、蛋白质 折叠、移位和组装，通过蛋白质-蛋白质作用 相互作用由ATP酶活性调节。 各种RNA和DNA病毒， 包括人类病原体，在其生命的不同阶段招募HSP 70 循环，或在病毒感染后诱导HSP 70。 但一般 蛋白伴侣在病毒-宿主相互作用中的作用是未知的。 甜菜 在本项目中使用的黄化梭状病毒（BYV）编码HSP 70同源物， 这是病毒在细胞间转移所必需的。 使用BYV作为 模型提供了前所未有的机会，直接探讨功能， HSP 70 h在病毒致病中的作用机制。 三个具体目标 该项目的主要内容是结构/功能关系，亚细胞 HSP 70 h表达的调控。 A.功能性解剖 HSP 70 h。 功能重要的结构元件，ATP酶的作用 HSP 70 h结构域的活性和特异性将被表征 利用反向遗传学和生物化学方法。 结构保护 细胞和病毒HSP 70之间的差异将用于设计突变， 引入全长BYV cDNA。 BYV标记的可用性 通过GUS或GFP报告基因将有助于突变体的功能研究 表型 B。HSP 70 h的蛋白质伴侣。 因为所有的细胞HSP 70 通过蛋白质-蛋白质相互作用进行操作，鉴定靶蛋白 对HSP 70 h作用机制的研究将为进一步阐明HSP 70 h的作用机制提供重要线索。 HSP 70 h的配偶体蛋白将使用亲和色谱法鉴定， 免疫组织化学和酵母双杂交提供的体内测定 系统 C. HSP 70 h的亚细胞分布及其调控 定位 感染细胞和组织中的HSP 70 h将通过免疫组织化学方法检测。 免疫组化方法。 此外，通过HSP 70 h-GFP融合物产生的HSP 70 h-GFP融合物 将利用经修饰的病毒来揭示HSP 70 h在活体中的分布 细胞 HSP 70 h的转录和翻译调控将被研究。 在使用重组BYV的原生质体转染实验中研究 在HSP 70 h启动子的控制下表达报告基因。

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.