FUNCTION OF HSP70 HOMOLOG ENCODED IN RNA VIRUS

描述：这个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 发病机制。细胞热休克蛋白70在应激反应中是至关重要的，蛋白质 折叠、移位和组装，通过蛋白质-蛋白质起作用 受ATPase活性调节的相互作用。潜水员RNA和DNA病毒， 包括人类病原体，在他们生命的不同阶段招募Hsp70 循环，或在病毒感染后诱导Hsp70。然而，将军 蛋白质伴侣在病毒-宿主相互作用中的作用尚不清楚。甜菜 本项目中使用的黄色闭锁病毒(BYV)编码一个HSP70同源基因 这对于病毒在细胞间的转移是至关重要的。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在病毒致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表达的分布和调控。A.功能性解剖 HSP70h。功能上重要的结构元件、ATPase的作用 将描述HSP70h结构域的活性和专门化 使用反向遗传和生化方法。结构保护 在细胞和病毒中，Hsp70将被用来设计突变， 将其导入到BYV全长cDNA中。标记的BYV的可用性 GUS或GFP记者将促进突变体的功能研究 表型。B.HSP70h的蛋白质伴侣。因为所有蜂窝Hsp70 通过蛋白质-蛋白质相互作用，识别目标蛋白质 HSP70h的表达将为HSP70h的作用机制提供重要线索。 HSP70h的配对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检测 系统。C.HSP70h的亚细胞分布及其调控。本土化 感染细胞和组织中热休克蛋白70h的含量将通过 免疫组织化学方法。此外，HSP70h-GFP融合由 将利用修饰的病毒揭示HSP70h在活体中的分布 细胞。HSP70h的转录和翻译调节将是 重组BYV原生质体转染法的研究 在HSP70h启动子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.