In vivo characterization of RNA virus assembly lines with EM tomography

使用 EM 断层扫描对 RNA 病毒装配线进行体内表征

• 批准号: 7659813
• 负责人: Jason Kenneth Lanman
• 金额: $ 9万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-25 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7659813
• 关键词: Abbreviations; Address; Affect; Area; Binding; Biochemical Genetics; Biological Preservation; Capsid; Capsid Proteins; Cell Culture Techniques; Cell membrane; Cells; Cellular Membrane; Chimera organism; Complex; Cryoelectron Microscopy; Cytoskeleton; Data; Development; Electron Microscope; Electron Microscopy; Environment; Excision; Facilities and Administrative Costs; Freeze Substitution; Freezing; Future; Genetic; Genome; Goals; Grant; Hemagglutinin; Housing; Human; Image; In Situ; In Vitro; Influenza; Label; Learning; Length; Location; Methods; Microscopy; Mitochondria; Modeling; Molecular; Mutation; Neuraminidase; Nucleoproteins; Organelles; Physiologic pulse; Positioning Attribute; Principal Investigator; Process; Protein Biosynthesis; Proteins; Protocols documentation; RNA; RNA Viruses; RNA replication; RNA-Directed RNA Polymerase; Relative (related person); Research Personnel; Resolution; Ribonucleoproteins; Ribosomes; Sampling; Societies; Staging; Structure; System; Testing; Three-Dimensional Imaging; Time; Tomogram; Training; United States; Viral; Virion; Virus; Virus Assembly; cost; human disease; in vivo; influenzavirus; mutant; novel; pandemic disease; particle; pressure; programs; protein structure; research study; skills; tomography; trafficking; viral RNA; virology

DESCRIPTION (provided by applicant): Virus assembly occurs in the complex three-dimensional environment of the cell, and new advances in electron microscopy tomography (EMT) now make it possible to construct high-resolution three-dimensional images where the spatial location of components involved in virus assembly can be determined. The simplicity of the Flock House virus (FHV) capsid and the extensive structural, biochemical, and genetic characterization of the virus make it an excellent system for studying in situ virus assembly. The focus of this proposal is to use to EMT to answer key questions about Flock House virus assembly in the cell and then extend these approaches to Influenza virus genome packaging and assembly. Aim 1: Three-dimensional imaging of the Flock House virus factories in vitro and in situ. Our hypothesis is FHV assembles in viral factories, which are formed from extensively modified mitochondria and are then trafficked on cytoskeleton to viral arrays. The modified mitochondria will be isolated and imaged using cryo-EMT to obtain near native state samples. The viral factories will then be imaged using cellular EMT to understand the virus assembly process in the context of the cell. Aim 2: EM-tomography analysis of influenza genome packaging. The Influenza virus genome is divided into eight RNA segments, and a long-standing question has been what is the mechanism for packaging all eight segments. Our hypothesis is that each of the eight viral RNAs (vRNA) are specifically packaged, and the removal of one vRNA will produce virus particles containing seven segments instead of eight. Virus particles missing a vRNA will be analyzed with EMT to determine the number of segments and which segments are packaged in each virus particle. Relevance: Viruses cause many human diseases throughout the world and result in major financial losses to society. In the United States Influenza is responsible for an estimated total direct and indirect cost of $10 billion per year, and the costs of a future pandemic could cost hundreds of billions of dollars. Investigating virus assembly and genome packaging will help researchers develop novel anti-viral targets.

描述（由申请人提供）：病毒组装发生在细胞的复杂三维环境中，电子显微镜断层扫描（EMT）的新进展使得可以构造高分辨率的三维图像，其中可以确定参与病毒组件的组件的空间位置。羊群病毒（FHV）衣壳的简单性以及该病毒的广泛结构，生化和遗传表征使其成为研究原位病毒组装的绝佳系统。该提案的重点是用于回答有关细胞中羊群病毒组装的关键问题，然后将这些方法扩展到流感病毒基因组包装和组装中。目标1：羊群房屋病毒工厂的三维成像在体外和原位。我们的假设是病毒工厂中的FHV组装，该病毒工厂是由广泛修饰的线粒体形成的，然后被贩运在细胞骨架上到病毒阵列。修改后的线粒体将通过冷冻EMT分离并成像，以获取近本天然状态样品。然后，将使用细胞EMT对病毒工厂进行成像，以理解细胞中的病毒组装过程。 AIM 2：流感基因组包装的EM-Tomography分析。流感病毒基因组分为八个RNA段，一个长期存在的问题是包装所有八个段的机制是什么。我们的假设是，八个病毒RNA（VRNA）中的每一个都是专门包装的，并且去除一个VRNA将产生包含七个片段而不是八个段的病毒颗粒。缺少VRNA的病毒颗粒将通过EMT分析，以确定每个病毒粒子中的片段数量和哪些段包装。 相关性：病毒导致世界上许多人类疾病，并对社会造成重大财务损失。在美国，流感估计每年的直接和间接成本为100亿美元，未来大流行的成本可能需要数亿美元。研究病毒组装和基因组包装将有助于研究人员开发新的抗病毒靶标。