Engineering host-pathogen interactions to understand influenza A infection

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.