Visualization of Influenza Viral RNA Assembly

甲型流感病毒（IAV）通过季节性流行和散发性大流行对公共卫生构成重大威胁。病毒基因组的分段性质促进重配，这是病毒之间的遗传物质在共感染细胞中交换的过程。在自然界中，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。从循环的人畜共患病毒种群中预测未来的大流行性流感病毒是困难的，因为对单个共感染细胞内的重配机制知之甚少。为了准确定义重组过程，我们必须首先了解细胞内病毒RNA（vRNA）组装的动力学。 流感病毒vRNA在细胞核中复制，并被运送到质膜进行包装，这需要所有八个片段的一个拷贝在单个病毒体中组装以产生完全感染性的病毒。 在该提案中，我们将基于我们先前关于流感组装的数据，并定义1）生理学相关的人和猪细胞类型中的组装动力学，2）调节vRNA转运的细胞蛋白，和3）共感染细胞内重配的位置。我们的中心假设是，vRNA装配发生在一个细胞类型的特定方式，与IAV在不同的宿主物种的重配。 本申请的特定目的将使用各种复杂的显微镜工具，包括使用定制光片显微镜进行活细胞成像，以确定各种细胞培养模型中的组装机制。 目的1利用荧光原位杂交和活细胞成像技术，探讨流感病毒vRNA在人和猪分化的气道上皮细胞中的组装动力学。 目的2将揭示流感病毒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，使用生物化学方法，如邻近依赖性生物素化。 目的3将联合收割机成像和基因组学方法相结合，以表征在分化的气道上皮细胞中与两种异源病毒共感染期间混合的vRNA的细胞定位。 拟议的工作将解决流感生物学中有关重配的许多悬而未决的问题，这些问题由于缺乏在生产性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.