Secreted mucus is a frontline defense against respiratory infection, enabling the capture and swift removal of infectious or irritating agents from the lungs. Airway mucus is composed of two mucins: mucin 5B (MUC5B) and 5AC (MUC5AC). Together, they form a hydrogel that can be actively transported by cilia along the airway surface. In chronic respiratory diseases, abnormal expression of these mucins is directly implicated in dysfunctional mucus clearance. Yet, the role of each mucin in supporting normal mucus transport remains unclear. Here, we generate human airway epithelial tissue cultures deficient in either MUC5B or MUC5AC to understand their individual contributions to mucus transport. We find that MUC5B and MUC5AC deficiency results in impaired and discoordinated mucociliary transport, respectively, demonstrating the importance of each mucin to airway clearance.

Rapid development of complex membranous replication structures is a hallmark of picornavirus infections. However, neither the mechanisms underlying such dramatic reorganization of the cellular membrane architecture, nor the specific role of these membranes in the viral life cycle are sufficiently understood. Here we demonstrate that the cellular enzyme CCTα, responsible for the rate-limiting step in phosphatidylcholine synthesis, translocates from the nuclei to the cytoplasm upon infection and associates with the replication membranes, resulting in the rerouting of lipid synthesis from predominantly neutral lipids to phospholipids. The bulk supply of long chain fatty acids necessary to support the activated phospholipid synthesis in infected cells is provided by the hydrolysis of neutral lipids stored in lipid droplets. Such activation of phospholipid synthesis drives the massive membrane remodeling in infected cells. We also show that complex membranous scaffold of replication organelles is not essential for viral RNA replication but is required for protection of virus propagation from the cellular anti-viral response, especially during multi-cycle replication conditions. Inhibition of infection-specific phospholipid synthesis provides a new paradigm for controlling infection not by suppressing viral replication but by making it more visible to the immune system. The cellular pathways hijacked to support viral replication may provide a promising class of targets for anti-viral therapeutics, which will be effective against broad groups of viruses relying on the same cellular pathways, and will likely be refractory to the development of resistance since cellular factors are not subject to selection. All (+)RNA viruses share the requirement for cellular membranes to assemble replication complexes. Here we investigated the mechanism underlying the massive membrane remodeling in poliovirus-infected cells. Our results demonstrate reorganization of the cellular lipid synthesizing machinery upon infection and identify lipid droplets as the organelles supporting the structural development of the replication membranes. Moreover, we show that inhibition of the infection-specific phospholipid synthesis renders virus propagation much more vulnerable to the cellular anti-viral defenses, providing a new direction for the development of anti-viral therapeutics.

Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus–host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air–liquid interface, organoids, or ‘on-chip’ technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium—the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.

In this study, influenza A virus (IAV) and nanoparticle diffusion in human airway mucus was quantified using fluorescent video microscopy and multiple particle tracking. In previous work, it was determined that mucin-associated sialic acid acts as a decoy receptor for IAV hemagglutinin binding, and that virus passage through the mucus gel layer is facilitated through the sialic-acid cleaving enzyme, neuraminidase (NA), also present on the IAV envelope. However, our data suggests the mobility of IAV in mucus is significantly influenced by the mesh structure of the gel, as measured by nanoparticle probes, and NA activity is not required to facilitate virus passage through mucus gels. Using newly developed analyses, the binding affinity of IAV to the 3D mucus meshwork was estimated for individual virions with dissociation constants in the mM range, indicative of weak and reversible IAV-mucus interactions. We also found IAV diffusion significantly increased in mucus when treated with a mucolytic agent to break mucin-mucin disulfide bonds. In addition, IAV diffusion was significantly limited in a synthetic mucus model as crosslink density was systematically increased and network pore size was reduced. The results of this work provide important insights on how the balance of adhesive and physical barrier properties of mucus influence the dissemination of IAV within the lung microenvironment.

Influenza A virus (IAV) causes seasonal epidemics and periodic pandemics, resulting in significant morbidity and mortality in the human population. Tethered mucin 1 (MUC1) is highly expressed in airway epithelium, the primary site of IAV replication, and also by other cell types that influence IAV infection, including macrophages. MUC1 has the potential to influence infection dynamics through physical interactions and/or signaling activity, and recent work suggests MUC1 acts as a releasable decoy receptor and anti-inflammatory molecule during IAV infection. Still, the modulation of MUC1 and its impact during viral pathogenesis remains unclear. Thus, we sought to further investigate the interplay between MUC1 and IAV in an in vitro model of primary human airway epithelium (HAE). Our data indicate that a recombinant IAV hemagglutinin (H3) and H3N2 virus can bind endogenous HAE MUC1. We find that infection of HAE cultures with H1N1 or H3N2 IAV strains does not trigger enhanced MUC1 shedding, but instead stimulates an increase in cell-associated MUC1 protein. We observed a similar increase after stimulation with either type I or type III interferon (IFN); however, inhibition of IFN signaling during H1N1 infection only partially abrogated this increase, indicating multiple soluble factors contribute to MUC1 upregulation during the antiviral response. We expanded these findings and demonstrate that in addition to HAE, primary human monocyte-derived macrophages also upregulate MUC1 protein in response to both IFN treatment and conditioned media from IAV-infected HAE cultures. We then developed HAE genetically depleted for MUC1 to determine its impact on IAV pathogenesis, finding that MUC1 knock-out cultures exhibited enhanced viral growth compared to control cultures for several IAV strains. Together, our data support a model whereby MUC1 antagonizes productive uptake of IAV in HAE. Infection then stimulates MUC1 expression on multiple cell types through IFN-dependent and -independent mechanisms that may further impact infection dynamics. Author Summary: The mucosal surface of the respiratory epithelium is an important site of first contact for viral respiratory pathogens. Large and heavily glycosylated molecules known as tethered mucins extend from the cell surface and may physically restrict access to underlying cells. Recently, one of these tethered mucins, MUC1, has also been shown to influence cell signaling and inflammation. Still, despite its abundance in the airway and multifunctional capability, the role of MUC1 during influenza virus infection in the human respiratory tract remains unclear. Here, we demonstrate that influenza virus directly interacts with MUC1 in a physiologically-relevant model of human airway epithelium and find that MUC1 protein expression is elevated throughout the epithelium and in primary human monocyte-derived macrophages in response to important antiviral signals produced during infection. Using genetically-modified human airway cultures lacking MUC1, we then provide evidence of more efficient influenza virus infection in the absence of this mucin. Our data suggest that MUC1 not only physically restricts influenza virus uptake, but also represents a dynamic component of the host response that acts to further stem viral spread.