Reovirus Factories: Structure, Function, and Dynamics

呼肠孤病毒工厂：结构、功能和动力学

• 批准号: 7333314
• 负责人: John S Parker
• 金额: $ 29.39万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333314
• 关键词: Address; Capsid; Capsid Proteins; Cells; Cytoplasm; Cytoplasmic Structures; Data; Development; Double-Stranded RNA; Electrophoresis; Excision; Fluorescence Microscopy; Genetic Transcription; Genome; Goals; Heat-Shock Proteins 70; Human; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Life; Lipids; Localized; Mammalian Orthoreovirus; Mass Spectrum Analysis; Mediating; Membrane; Membrane Lipids; Microinjections; Microscopy; Modeling; Molecular Chaperones; Movement; New Agents; Nucleosome Core Particle; Outcome; Play; Process; Proteins; Proteomics; RNA Interference; Reovirus; Research Proposals; Role; Structure; System; Techniques; Testing; Ubiquitin; Ubiquitination; Viral; Viral Genome; Viral Proteins; Virion; Virus; base; cancer therapy; inhibitor/antagonist; innovation; mitochondrial membrane; molecular dynamics; multicatalytic endopeptidase complex; mutant; novel; obligate intracellular parasite; positional cloning; prevent; research study; time use; two-dimensional

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.

描述（由申请人提供）：呼肠孤病毒是研究基础病毒病理生物学的模型药物，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（VF）的细胞质结构内复制和组装。这项提案的长期目标是了解细胞和病毒因子如何在VF中相互作用以调节病毒组装和复制。基于我们的初步研究结果，我们假设呼肠孤病毒病毒粒子在VF内的组装需要细胞蛋白，并且通过VF基质的重塑来调节。具体目标是： 1.鉴定与VF相关的细胞蛋白及其在病毒组装和/或 复制的与VF相关的细胞蛋白将被纯化并通过二维电泳分析。 电泳和质谱。将通过免疫荧光（IF）显微镜检查确认候选蛋白与感染细胞中VF的相关性。然后使用RNA干扰对选定的蛋白质进行功能分析，以确定它们在病毒复制过程中的需求， 2.确定HSP 70分子伴侣在组装外衣壳穆尔：sigma 3中的作用 异源六聚体和调节它们向VF的募集。将使用显性干扰hsc 70突变体和显微注射来测试伴侣在异源六聚体组装及其向VF募集中的功能作用。 实验 3.确定VF基质的重塑在呼肠孤病毒病毒粒子组装中的作用。蛋白酶体抑制剂对VF基质重塑和病毒体组装的功能影响将得到解决。抑制蛋白酶体降解和伴侣作用对VF运动的影响以及VF的分子动力学？将使用延时IF显微镜和基于荧光的实验评估活细胞中的NS并将其与病毒体组装相关联，以评估VF基质蛋白的扩散迁移率。