In vivo characterization of RNA virus assembly lines with EM tomography

描述(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)的新进展现在使构建高分辨率三维图像成为可能，其中涉及病毒组装的组件的空间位置可以确定。鸡舍病毒(FHV)衣壳的简单性以及病毒广泛的结构、生化和基因特征使其成为研究病毒原位组装的极佳系统。这项提议的重点是使用TO 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：体外和原位对鸡舍病毒工厂的三维成像。我们的假设是FHV在病毒工厂中组装，这些线粒体由广泛修改的线粒体形成，然后通过细胞骨架被贩运到病毒阵列。修改后的线粒体将被分离并使用冷冻电子显微镜成像，以获得接近自然状态的样本。然后将使用细胞EMT对病毒工厂进行成像，以了解病毒在细胞环境中的组装过程。目的2：流感基因组包装的电子断层扫描分析。流感病毒基因组被分成八个RNA片段，一个长期存在的问题是，将这八个片段打包的机制是什么。我们的假设是，八个病毒RNA(VRNA)中的每一个都是专门包装的，移除一个vRNA将产生包含七个片段的病毒颗粒，而不是八个片段。缺少vRNA的病毒颗粒将用EMT进行分析，以确定每个病毒颗粒中包装的片段数量和哪些片段。 相关性：病毒在世界各地导致许多人类疾病，并给社会造成重大经济损失。在美国，流感每年造成的直接和间接成本估计为100亿美元，未来大流行的成本可能高达数千亿美元。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点。

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.