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）：蠕虫病毒是研究基本病毒病理学的模型药物，并正在发展为令人兴奋的人类癌症治疗的新药物。静脉病毒在称为病毒工厂（VFS）的细胞质结构内复制并组装。该提案的长期目标是了解细胞和病毒因素如何在VF中相互作用以调节病毒组装和复制。基于我们的初步发现，我们假设在VF中组装葡萄病毒病毒体需要细胞蛋白，并通过重塑VF基质来调节。具体目的是： 1。鉴定与VF相关的细胞蛋白及其在病毒组装中的功能和/或 复制。与VF相关的细胞蛋白将通过二维纯化和分析 电泳和质谱。候选蛋白与受感染细胞中VF的缔合将通过免疫荧光（IF）显微镜证实。然后，选定的蛋白质将在功能上分析其在使用RNA干扰的病毒复制过程中的要求， 2。确定Hsp70伴侣在组装外带帽MUL中的作用：Sigma3 杂聚糖并监管VFS的招聘。伴侣在异己组装中的功能作用及其募集到VFS的功能将使用主要干扰HSC70突变体和显微注射测试 实验。 3。确定VF矩阵在依肠病毒病毒体组装中扮演重塑的作用。蛋白酶体抑制剂对VF基质重塑和病毒体组装的功能作用将得到解决。如果基于显微镜和基于荧光的实验评估VF基质蛋白的扩散性，则将评估蛋白酶体降解和伴侣作用对活细胞中VF的运动以及活细胞中的分子动力学的影响。