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: 8468182
• 负责人: Jason Kenneth Lanman
• 金额: $ 20.42万
• 依托单位: PURDUE UNIVERSITY
• 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 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8468182
• 关键词: Address; Biochemical Genetics; Capsid; Cells; Complex; Cytoskeleton; Development; Direct Costs; Electron Microscopy; Environment; Excision; Facilities and Administrative Costs; Future; Genome; Goals; Housing; Image; In Situ; Influenza; Instruction; Location; Microscopy; Mitochondria; Molecular; Positioning Attribute; Process; RNA; RNA Viruses; Research Personnel; Resolution; Societies; System; Three-Dimensional Imaging; Time; Tomogram; United States; Viral; Virion; Virus; Virus Assembly; cost; human disease; in vivo; influenzavirus; novel; pandemic disease; reconstruction; tomography; trafficking; viral RNA

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 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 viral factories will be imaged using EMT to understand the virus assembly process around the modified mitochondria and entire infected cells will reconstructed to investigate the extensive morphological changes that occur during the virus lifecycle. 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.

病毒组装发生在细胞的复杂三维环境中，并在 电子显微镜断层扫描（EMT）现在可以构建高分辨率三维 可以确定参与病毒组装的组件的空间位置的图像。这 羊群病毒（FHV）帽子的简单性以及广泛的结构，生化和遗传 病毒的表征使其成为研究原位病毒组装的绝佳系统。重点 建议是用来回答有关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：原位羊群病毒工厂的三维成像。 我们的假设是病毒工厂中的FHV组装，它们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。病毒工厂将使用EMT对 了解修饰的线粒体周围的病毒组装过程，整个受感染的细胞将 重建以研究病毒生命周期期间发生的广泛形态变化。 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片段，一个长期存在的问题是 包装所有八个细分市场的机制是什么？我们的假设是八个病毒RNA中的每一个 （VRNA）是专门包装的，去除一个VRNA将产生包含七个的病毒颗粒 细分而不是八个。缺少VRNA的病毒颗粒将通过EMT分析，以确定 每个病毒颗粒中包装的段数和哪些段的数量。