Engineering host-pathogen interactions to understand influenza A infection

像流感这样的呼吸道病毒已经进化到可以绕过人体对感染的自然防御。吸入肺部后，病毒颗粒必须穿过覆盖并保护肺部的粘液。然而，目前还没有完全了解是什么让这些病毒绕过气道粘液到达底层细胞。该项目旨在了解流感病毒的物理大小和形状如何影响其克服粘液屏障的能力。此外，将设计研究以确定这些病毒如何偏好结合粘液中和气道上皮细胞中丰富的某些糖，从而影响它们在粘液中的运动。这项工作将为未来研究其他呼吸道病原体，如鼻病毒，呼吸道合胞病毒（RSV）和冠状病毒（例如，SARS-CoV-2）。 该项目将为来自不同背景的研究生和本科生提供多学科培训机会。 本项目的目标是了解影响甲型流感病毒（IAV）穿透粘膜屏障感染能力的病毒和宿主因素。粘液由高度糖基化的粘蛋白质组成，并在肺微环境中对病原体提供生物化学和物理屏障。为了引发感染，甲型流感病毒（IAV）以菌株依赖性受体偏好结合气道上皮表面上的α 2，3-和/或2，6-连接的唾液酸。IAV对唾液酸糖型的偏好可能会改变病毒与粘蛋白相关唾液酸的相互作用。然而，目前还不清楚这可能如何影响粘液对IAV的捕获。此外，IAV天然产生具有球形和丝状形状的病毒体。虽然已经探索了IAV感染中的其他病毒体相关因素，但IAV形态学的作用仍不清楚。在宿主方面，由于有效模拟粘膜抗病毒防御的挑战，粘液的屏障功能及其对IAV感染的影响尚不清楚。为了详细研究这些复杂的宿主-病原体相互作用，该项目将使用荧光视频显微镜和多粒子跟踪图像分析来测量从人支气管上皮培养物和生物工程合成粘液中收集的粘液中IAV粒子的扩散。所采取的方法将结合分子病毒学来产生具有特定受体偏好和形状的IAV颗粒，以及生物化学工程方法来调节合成粘液屏障中的聚糖表达。这个全面的工具箱将用于分析IAV渗透是病毒体形态和聚糖结合偏好以及粘液屏障的组成和结构的函数的假设。定量检测IAV-粘液相互作用的独特能力使得这种方法非常有价值，并且是对标准感染性测定的补充。在这项基础工作的基础上，将创建一种新型的高通量病毒感染性筛选系统，该系统将解释粘液屏障特性的影响。通过这项工作开发的独特的、高度创新的工具将增强我们对IAV的理解，并为影响病毒在感染前如何通过粘液的因素提供新的视角。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Respiratory viruses like influenza have evolved to circumvent the body’s natural defenses to infection. Following inhalation into the lung, viral particles must navigate through mucus that coats and protects that lung. However, it is not yet fully understood what allows these viruses to bypass airway mucus to reach underlying cells. This project seeks to understand how the physical size and shape of influenza virus influences its ability to overcome the mucus barrier. In addition, studies will be designed to determine how the preference of these viruses to bind to certain sugars found in abundance within mucus and on airway epithelial cells impacts their movement within mucus. This work will lay the foundation for future studies on other respiratory pathogens such as rhinovirus, respiratory syncytial virus (RSV), and coronaviruses (e.g., SARS-CoV-2). The project will provide multidisciplinary training opportunities for graduate and undergraduate students who will be actively recruited from diverse backgrounds. The goal of this project is to understand both the viral and host factors that influence the ability of Influenza A virus (IAV) to penetrate the mucosal barrier to infection. Mucus is composed of heavily glycosylated mucin proteins and presents both a biochemical and physical barrier to pathogens within the lung microenvironment. To initiate infection, influenza A virus (IAV) binds to alpha 2,3- and/or 2,6-linked sialic acid on the airway epithelial surface with a strain-dependent receptor preference. The preference of IAV for sialic acid glycoforms may alter virus interactions with mucin-associated sialic acid. Yet, it is unknown how this may influence trapping of IAV by mucus. Furthermore, IAV naturally produces virions with both spherical and filamentous shape. While other virion-associated factors in IAV infection have been explored, the role of IAV morphology remains unclear. On the host side, barrier function of mucus and its impact on IAV infection are not understood due to the challenges in effectively modeling mucosal antiviral defense. To examine these complex host-pathogen interactions in detail, the project will measure diffusion of IAV particles in mucus collected from human bronchial epithelial cultures and bioengineered synthetic mucus using fluorescent video microscopy and multiple particle tracking image analysis. The approach taken will incorporate molecular virology to produce IAV particles with specified receptor preference and shape as well as biochemical engineering methods to modulate glycan expression in synthetic mucus barriers. This comprehensive toolbox will be used to analyze the hypothesis that IAV penetration is a function of virion morphology and glycan-binding preferences as well as the composition and architecture of the mucus barrier. The unique ability to quantitatively examine IAV-mucus interactions makes this approach highly valuable and complementary to standard assays of infectivity. Building on this fundamental work, a novel high-throughput viral infectivity screening system that accounts for the impact of mucus barrier properties will be created. The unique, highly innovative tools developed through this work will enhance our understanding of IAV and offer a new perspective on the factors that influence how viruses pass through mucus prior to infection.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.