In vivo characterization of RNA virus assembly lines with EM tomography

病毒组装发生在细胞的复杂三维环境中，在 电子显微镜层析成像(EMT)使构建高分辨率的三维图像成为可能 可以确定病毒组装中涉及的组件的空间位置的图像。这个 鸡舍病毒衣壳的简单性及其广泛的结构、生化和遗传 病毒的特性使其成为研究病毒原位组装的极佳系统。这件事的重点是 建议使用EMT来回答有关鸡舍病毒在细胞内组装的关键问题，然后 将这些方法扩展到流感病毒基因组包装和组装。 目的1：现场对鸡舍病毒工厂进行三维成像。 我们的假设是FHV在病毒工厂中组装，这些工厂是由广泛修改的线粒体形成的 然后通过细胞骨架被贩卖到病毒阵列。病毒工厂将使用EMT进行成像 了解病毒围绕修饰的线粒体和整个感染细胞的组装过程 重建以研究在病毒生命周期中发生的广泛的形态变化。 目的2：流感基因组包装的电子断层扫描分析。 流感病毒基因组分为八个RNA片段，一个长期存在的问题是 包装所有八个细分市场的机制是什么？我们的假设是，八种病毒RNA中的每一种 (VRNA)是专门包装的，移除一个vRNA将产生包含七个vRNA的病毒颗粒 分段而不是八段。缺失vRNA的病毒颗粒将用EMT进行分析，以确定 每个病毒粒子中包含的片段数以及打包的片段。

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.