Genetic and biophysical mechanisms that control influenza virus cellular multiplicity of infection

被流感感染的单个细胞可以产生数百到数千个具有传染性的新病毒粒子。这些病毒粒子不均匀地传播，在每个细胞中产生广泛的病毒载量变化，这些病毒载量集中在感染的初始部位周围。感染特定细胞的病毒数量的差异会影响该细胞是否产生自己的新病毒粒子，或者是否产生抗病毒反应。因此，了解流感病毒如何传播对于了解感染如何进展以及宿主如何反应至关重要。该项目的中心目标是了解病毒和宿主的遗传和生物物理特征如何影响流感病毒细胞传播的空间结构，以及细胞传播的差异如何影响感染的进展和由此产生的细胞反应。我们先前的数据表明，流感的遗传和生物物理特征控制着病毒在细胞水平上的传播方式。这些特征与三种病毒蛋白密切相关：HA、NA和M1。受体结合蛋白HA介导病毒附着naïve细胞，而受体破坏蛋白NA促进病毒释放和传播。基质蛋白M1控制病毒颗粒的形状以及HA和NA在病毒粒子表面的分布。总的来说，这些蛋白质控制着病毒颗粒的生物物理特性，并塑造了病毒粒子在宿主体内传播的方式。我们假设，通过这些蛋白的遗传机制，以及参与黏毛清除的宿主因子，决定了病毒传播的空间模式和细胞共感染的频率，从而塑造了疾病的进展。我们将通过两个具体目标来检验这一假设。在目标1中，我们将使用高分辨率成像来跟踪病毒粒子的传播和病毒感染，我们将确定这如何依赖于HA， NA和M1的自然变化。通过这些实验，我们将确定这些蛋白质如何共同影响多周期病毒复制过程中发生的细胞共感染程度。在Aim 2中，我们将研究参与粘膜纤毛清除的宿主因素如何促进IAV的细胞传播，我们将确定病毒和宿主因素的集体影响，这些因素改变了共同感染的频率，对分化的人气道细胞的关键感染结果。该项目的预期结果是提高对流感病毒表面和结构蛋白如何通过调整多周期生长过程中发生的共感染程度来促进病毒复制的细胞内方面的理解。从这项工作中获得的见解将有助于了解流感病毒如何在宿主环境中导航，并将识别导致有时观察到的不同感染结果的宿主和病毒因素。该提案还将引入新的工具和方法来研究流感病毒感染的空间组织和动态。

Single cells infected by influenza can produce hundreds to thousands of infectious new virions. These virions spread non-uniformly, producing wide variations in the viral load per cell that are concentrated around the initial site of infection. Differences in the amount of virus that infects a particular cell can influence whether or not that cell produces new virions of its own, or if it mounts an anti-viral response. Understanding how influenza virions spread is therefore critical to understanding how infection progresses and how the host responds. The central goal of this project is to understand how genetic and biophysical features of both virus and host contribute to the spatial structure of influenza virus cellular spread, and how differences in cellular spread shape the progression of infection and the resulting cellular responses. Our prior data demonstrate that genetic and biophysical features of influenza control the way that the virus spreads at the cellular level. These features are strongly linked to three viral proteins in particular: HA, NA, and M1. The receptor-binding protein HA mediates virus attachment to naïve cells, while the receptor-destroying protein NA facilitates virus release and dissemination. The matrix protein M1 controls the shape of the virus particle and the distribution of HA and NA on the virion surface. Collectively, these proteins control the biophysical characteristics of virus particles and shape the way that virions spread throughout the host. We hypothesize that genetic mechanisms acting through these proteins, together with host factors involved in mucociliary clearance, determine the spatial pattern of viral spread and the frequency of cellular co-infection, thereby shaping the progression of disease. We will test this hypothesis through two specific aims. In Aim 1, we will use high- resolution imaging to track the spread of virions and viral infection, and we will determine how this depends on natural variations in HA, NA, and M1. Through these experiments, we will identify how these proteins collectively influence the degree of cellular co-infection that occurs during multi-cycle virus replication. In Aim 2, we will investigate how host factors involved in mucociliary clearance contribute to cellular spread of IAV, and we will determine the collective impact of viral and host factors that alter the frequency of co-infection on key infection outcomes in differentiated human airway cells. The expected outcome of this project is an improved understanding of how influenza virus surface and structural proteins contribute to intracellular aspects of viral replication by tuning the degree of co-infection that occurs during multi-cycle growth. Insights from this work will inform basic understanding of how influenza viruses navigate the host environment and will identify host and viral factors that contribute to the disparate outcomes of infection that are sometimes observed. This proposal will also introduce new tools and methodologies for investigating the spatial organization and dynamics of influenza virus infection.