Reovirus Factories: Structure, Function, and Dynamics

描述（由申请人提供）：呼肠孤病毒是研究基本病毒病理生物学的模型药物，并正在发展成为令人兴奋的人类癌症治疗新药物。呼肠孤病毒在称为病毒工厂（VFs）的细胞质结构内复制和组装。本提案的长期目标是了解细胞和病毒因子如何在VFs内相互作用以调节病毒组装和复制。基于我们的初步发现，我们假设呼肠孤病毒病毒体在VF内的组装需要细胞蛋白，并通过VF基质的重塑来调节。具体目标是：

DESCRIPTION (provided by applicant): Reoviruses are model agents for studying basic viral pathobiology and are developing into exciting new agents for human cancer therapy. Reoviruses replicate and assemble within cytoplasmic structures called viral factories (VFs). The long-term goals of this proposal are to understand how cellular and viral factors interact within VFs to regulate viral assembly and replication. Based on our preliminary findings, we hypothesize that assembly of reovirus virions within VFs requires cellular proteins and is regulated by remodeling of the VF matrix. The specific aims are: 1. To identify cellular proteins associated with VFs and their function(s) in viral assembly and/or replication. Cellular proteins associated with VFs, will be purified and analyzed by 2-dimensional electrophoresis and mass spectrometry. Association of candidate proteins with VFs in infected cells will be confirmed by immunofluorescence (IF) microscopy. Selected proteins will then be functionally analyzed for their requirement during viral replication using RNA interference, 2. To identify the role(s) of HSP70 chaperones in assembling outer capsid mul:sigma3 heterohexamers and in regulating their recruitment to VFs. The functional role of chaperones in assembly of heterohexamers and their recruitment to VFs will be tested using dominant interfering hsc70 mutants and microinjection experiments. 3. To determine the role that remodeling of the VF matrix plays in assembly of the reovirus virion. The functional effects of proteasomal inhibitors on VF matrix remodeling and assembly of virions will be addressed. The effects of inhibiting proteasomal degradation and chaperone action on the movement of VFs and the molecular dynamics of ?NS in living cells will be assessed and correlated with virion assembly using time-lapse IF microscopy and fluorescence-based experiments to assess the diffusional mobilities of the VF matrix protein.