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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

基本信息

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
项目类别：
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

项目摘要

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复杂的三维环境中，电子显微镜断层扫描（EMT）的新进展现在使构建高分辨率三维图像成为可能，其中可以确定病毒组装中涉及的组件的空间位置。禽舍病毒（FHV）衣壳的简单性和病毒广泛的结构、生化和遗传特征使其成为研究病毒原位组装的一个极好的系统。本提案的重点是使用EMT来回答有关禽舍病毒在细胞中组装的关键问题，然后将这些方法扩展到流感病毒基因组的包装和组装。目的1：禽舍病毒工厂的体外和原位三维成像。我们的假设是FHV在病毒工厂中组装，病毒工厂由广泛修饰的线粒体形成，然后通过细胞骨架运输到病毒阵列。修饰的线粒体将被分离并使用冷冻emt成像以获得接近天然状态的样品。然后将使用细胞EMT对病毒工厂进行成像，以了解病毒在细胞背景下的组装过程。目的2：流感基因组包装的电子扫描断层扫描分析。流感病毒基因组被分成八个RNA片段，一个长期存在的问题是包装这八个片段的机制是什么。我们的假设是，8个病毒rna （vRNA）中的每一个都被专门包装，去除一个vRNA将产生含有7个片段而不是8个片段的病毒颗粒。缺失vRNA的病毒颗粒将使用EMT进行分析，以确定每个病毒颗粒中包含的片段数量和哪些片段。