Visualization of Influenza Viral RNA Assembly

甲型流感病毒(IAV)通过季节性流行和零星流行对公共卫生构成重大威胁。病毒基因组的节段性促进了重新排序，这是一种在病毒之间的遗传物质在共同感染的细胞中交换的过程。在自然界，重新分类会导致病毒多样性的增加和大流行性流感病毒的出现。例如，2009年甲型H1N1流感大流行病毒是由两种流行的猪病毒重新组合而成的。从流行的人畜共患病病毒种群中预测未来的大流行流感病毒是困难的，因为人们对单个共同感染细胞内的重新分类机制知之甚少。为了准确地定义重组的过程，我们必须首先了解细胞内病毒RNA(VRNA)组装的动力学。流感vRNA在细胞核内复制，并被运输到质膜进行包装，这需要所有八个片段的一个副本在单个病毒粒子中组装，以产生完全具有感染性的病毒。在这项提案中，我们将以我们以前关于流感组装的数据为基础，定义1)生理相关的人和猪细胞类型的组装动力学，2)调节vRNA运输的细胞蛋白，以及3)共同感染细胞内的重组位置。我们的中心假设是vRNA组装以一种细胞类型特定的方式发生，这种方式与不同宿主物种中的IAV重排相关。该应用程序的具体目标将使用各种复杂的显微镜工具，包括带有定制光片显微镜的实时细胞成像，以确定各种细胞培养模型中的组装机制。目的1利用多色荧光原位杂交和活细胞成像技术，探索流感病毒vRNA在人和猪分化的呼吸道上皮细胞中组装的动力学。目的2将揭示细胞细胞骨架蛋白和膜细胞器在流感vRNA组装过程中使用的生物化学方法的身份和作用，如邻近依赖的生物素化。目的3将结合成像和基因组学方法来表征vRNA在与两种异源病毒在分化的呼吸道上皮细胞中混合感染时的细胞位置。拟议的工作将解决流感生物学中许多悬而未决的问题，这些问题是由于缺乏工具来跟踪生产性感染期间活细胞中的vRNA运动而仍未得到回答的。此外，这些研究将确定vRNA包装中涉及的新宿主因子，可作为潜在的治疗靶点。

Influenza A viruses (IAV) pose a major public health threat through both seasonal epidemics and sporadic pandemics. The segmented nature of the viral genome promotes reassortment, a process where the genetic material between viruses is exchanged in a co-­infected cell. In nature, reassortment leads to increased viral diversity and emergence of pandemic influenza viruses. For example, the 2009 influenza H1N1 (‘swine flu’) pandemic virus, emerged from reassortment of two circulating swine viruses. Prediction of future pandemic influenza viruses from circulating zoonotic virus populations is difficult because very little is known about the mechanism of reassortment within a single co-infected cell. To accurately define the process of reassortment, we must first understand the dynamics of intracellular viral RNA (vRNA) assembly. Influenza vRNA replicates in the nucleus and is transported to the plasma membrane for packaging, which requires one copy of all eight segments to assemble within a single virion to produce a fully infectious virus. In this proposal, we will build upon our previous data on influenza assembly and define 1) the assembly dynamics in physiologically relevant human and swine cell types, 2) the cellular proteins modulating vRNA transport, and 3) the location of reassortment within a co-­infected cell. Our central hypothesis is that vRNA assembly occurs in a cell-­type specific manner that correlates with IAV reassortment in different host species. The Specific Aims of this application will use a variety of sophisticated microscopy tools, including live cell imaging with a custom light-­sheet microscope, to determine the assembly mechanism in various cell culture models. Aim 1 will utilize multicolor fluorescent in situ hybridization and live cell imaging techniques to explore the dynamics of influenza vRNA assembly in human and swine differentiated airway epithelial cells. Aim 2 will uncover the identity and roles of cellular cytoskeletal proteins and membranous organelles utilized during influenza vRNA assembly using biochemical approaches like proximity-­dependent biotinylation. Aim 3 will combine imaging and genomic approaches to characterize the cellular location of vRNA intermingling during co-infection with two heterologous viruses in differentiated airway epithelial cells. The proposed work will address many outstanding questions in influenza biology regarding reassortment that have remained unanswered due to a lack of tools to track vRNA movement in live cells during a productive infection. In addition, these studies will identify novel host factors involved in vRNA packaging that can be pursued as potential therapeutic targets.