Reovirus Factories: Structure, Function, and Dynamics

描述(申请人提供)：呼肠孤病毒是研究基础病毒病理生物学的模型制剂，正在发展成为人类癌症治疗的令人兴奋的新制剂。呼肠孤病毒在称为病毒工厂(VFS)的细胞质结构中复制和组装。这项提议的长期目标是了解细胞和病毒因子如何在VFS中相互作用，以调节病毒的组装和复制。根据我们的初步发现，我们假设呼肠病毒病毒粒子在VFS内的组装需要细胞蛋白，并受VF基质的重塑调节。具体目标是： 1.鉴定与VFS相关的细胞蛋白及其在病毒组装和/或中的功能(S) 复制。与VFS相关的细胞蛋白将被纯化并进行二维分析 电泳法和质谱法。免疫荧光(IF)显微镜将证实感染细胞中的候选蛋白与VFS的相关性。然后将使用RNA干扰对选定的蛋白质在病毒复制过程中的需求进行功能分析， 2.确定热休克蛋白70分子伴侣在组装外衣壳中的作用(S) 异六角体和规范他们被招募到越南船级社。将使用显性干扰hsc70突变体和显微注射来测试伴侣在组装异六聚体中的功能作用以及它们在VFS中的募集。 实验。 3.确定VF基质重塑在呼肠孤病毒粒子组装中的作用。蛋白酶体抑制剂在VF基质重构和病毒粒子组装中的功能作用将被讨论。抑制蛋白酶体降解和伴侣作用对VFS运动的影响和活细胞中NS的分子动力学将被评估，并与病毒粒子组装相关联，使用时间推移IF显微镜和基于荧光的实验来评估VF基质蛋白的扩散迁移率。

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.