Visualization of Influenza Viral RNA Assembly

甲型流感病毒（IAV）通过季节性流行和散发大流行对公共卫生构成重大威胁。病毒基因组的片段性促进了重组，这是一个病毒之间的遗传物质在共感染细胞中交换的过程。在本质上，重组导致病毒多样性增加和大流行性流感病毒的出现。例如，2009年H1N1流感（“猪流感”）大流行病毒是由两种流行的猪病毒重组而成的。由于对单个共感染细胞内的重配机制知之甚少，因此很难从流行的人畜共患病毒群体中预测未来的大流行性流感病毒。为了准确地定义重组过程，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，并被运送到质膜进行包装，这需要在单个病毒粒子内组装所有八个片段的一个副本，以产生完全感染性的病毒。在本提案中，我们将基于我们之前关于流感组装的数据，并定义1)生理上相关的人类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)共感染细胞内重组的位置。我们的中心假设是，vRNA组装以细胞类型特异性的方式发生，与不同宿主物种的IAV重组相关。该应用程序将使用各种复杂的显微镜工具，包括使用定制光片显微镜的活细胞成像，以确定各种细胞培养模型中的组装机制。目的1将利用多色荧光原位杂交和活细胞成像技术来探索流感病毒vRNA在人和猪分化的气道上皮细胞中的组装动力学。目的2将揭示在流感vRNA组装过程中使用的细胞骨架蛋白和膜细胞器的身份和作用，如邻近依赖的生物素化。目的3将结合影像学和基因组学方法来表征分化气道上皮细胞中两种异源病毒共感染时vRNA混杂的细胞位置。这项工作将解决流感生物学中关于重组的许多悬而未决的问题，这些问题由于缺乏在生产感染期间跟踪活细胞中vRNA运动的工具而仍未得到解答。此外，这些研究将确定参与vRNA包装的新宿主因子，这些因子可以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.