In vivo characterization of RNA virus assembly lines with EM tomography

病毒组装发生在细胞复杂的三维环境中，新进展 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。的 Flock House 病毒 (FHV) 衣壳的简单性和广泛的结构、生化和遗传 病毒的表征使其成为研究原位病毒组装的优秀系统。本次的重点 建议利用 EMT 来回答有关 Flock House 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包装和组装。 目标 1：Flock House 病毒工厂原位三维成像。 我们的假设是 FHV 在病毒工厂中组装，病毒工厂由经过广泛修饰的线粒体形成 然后通过细胞骨架运输至病毒阵列。病毒工厂将使用 EMT 进行成像 了解修饰后的线粒体和整个受感染细胞周围的病毒组装过程 重建以研究病毒生命周期中发生的广泛形态变化。 目标 2：流感基因组包装的电磁断层扫描分析。 流感病毒基因组分为八个RNA片段，一个长期存在的问题是 包装所有八个段的机制是什么？我们的假设是八种病毒 RNA 中的每一种 (vRNA)经过专门包装，去除1个vRNA就会产生含有7个vRNA的病毒颗粒 段而不是八段。缺少 vRNA 的病毒颗粒将通过 EMT 进行分析，以确定 片段的数量以及每个病毒颗粒中包装了哪些片段。

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.