Hsp70 as Dispatcher of Virus Assembly and Trafficking

描述（由申请人提供）：人类、动物和植物病毒的感染周期包括病毒粒子在受感染生物体内的组装和传播过程。这些过程需要特殊的病毒衣壳和转运蛋白的功能，以及病毒招募的细胞蛋白和细胞器的功能。在病毒粒子形成和传播过程中，病毒和宿主组分之间的功能协调机制了解相对较少。该项目开发了甜菜黄病毒（BYV）模型，利用创新的生化、遗传和细胞生物学方法揭示病毒与细胞蛋白之间的相互作用。发现Hsp70分子伴侣的BYV同源物（Hsp70h）通过细胞结构的修饰整合了病毒粒子组装和运输的主要功能。BYV模型为研究这些过程提供了独特的优势，包括在感染细胞内可视化病毒与宿主相互作用的能力。

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.