Hsp70 as Dispatcher of Virus Assembly and Trafficking

描述（由申请方提供）：人、动物和植物病毒的感染周期包括病毒体在受感染生物体内的组装和传播过程。这些过程需要专门的病毒衣壳和转运蛋白，以及由病毒募集的细胞蛋白和细胞器的功能。在病毒体形成和传播过程中，病毒和宿主成分之间的功能协调了解相对较少。甜菜黄化病毒（BYV）模型是在该项目期间开发的，以利用创新的生物化学、遗传学和细胞生物学方法揭示病毒和细胞蛋白之间的相互作用。发现BYV Hsp 70分子伴侣同源物（Hsp 70 h）整合了病毒体组装和运输的主要功能，并改变了细胞结构。BYV模型为研究这些过程提供了独特的优势，包括能够可视化活感染细胞内的病毒-宿主相互作用。 两个具体目标是这项建议的重点。目的1是确定BYV病毒粒子的分子结构。这些病毒粒子拥有尾巴，作为Hsp 70 h授权的专门运动装置。尾部蛋白的拓扑结构将使用电子和原子力显微镜来解析。将使用可用的足底模型解剖尾部组件的HSP 70 h引导机制。目的二是研究热休克蛋白70 h与宿主细胞的相互作用。Hsp 70 h与宿主蛋白相互作用以重塑细胞环境并促进与细胞骨架运动系统相关的病毒运输的假设将被测试。使用BYV变异体，表达细胞器靶向的荧光报告，实时变化的细胞结构在病毒感染期间将被检查。将使用酵母双杂交筛选和质谱分析鉴定与Hsp 70 h相互作用的宿主蛋白。将使用靶向Hsp 70 h和宿主蛋白的遗传策略来评估所观察到的相互作用的功能意义。

DESCRIPTION (provided by applicant): Infection cycles of the human, animal, and plant viruses include processes of virion assembly and dissemination within the infected organism. These processes require functions of the specialized viral capsid and transport proteins, as well as cellular proteins and organelles recruited by virus. The functional coordination among viral and host components during virion formation and spread are understood relatively poorly. The beet yellows virus (BYV) model was developed during this project to reveal interactions between the viral and cell proteins using innovative biochemical, genetic and cell biological approaches. The BYV homolog of Hsp70 molecular chaperones (Hsp70h) was found to integrate principal functions of virion assembly and transport with modifications of the cell architecture. The BYV model provides unique advantages for investigation of these processes, including ability to visualize virus-host interactions within the live infected cells. Two Specific Aims are in the focus of this proposal. Aim 1 is designed to determine the molecular architecture of the BYV virions. These virions possess the tails, which serve as specialized movement devices empowered by Hsp70h. The topology of the tail proteins will be resolved using electron and atomic force microscopy. The Hsp70h-guided mechanism of the tail assembly will be dissected using available in planta model. Aim 2 will address the interactions between Hsp70h and the host cell. The hypothesis stating that Hsp70h interacts with host proteins to remodel cellular environment and facilitate virus trafficking in association with the cytoskeletal motility systems will be tested. Using BYV variants that express organelle-targeted fluorescent reporters, real-time changes in cell architecture during virus infection will be examined. Host proteins that interact with Hsp70h will be identified using yeast two-hybrid screens and mass spectrometry analyses. Functional significance of the observed interactions will be assessed using genetic strategies targeting both Hsp70h and host proteins.