Membrane-Tethered Mucin 1 Is Stimulated by Interferon and Virus Infection in Multiple Cell Types and Inhibits Influenza A Virus Infection in Human Airway Epithelium.

Influenza A virus (IAV) causes 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, yet MUC1 modulation and its impact during viral pathogenesis remain unclear. Thus, we investigated MUC1-IAV interactions in an in vitro model of human airway epithelium (HAE). Our data indicate that a recombinant IAV hemagglutinin (H3) and H3N2 virus can bind endogenous HAE MUC1. Notably, infection of HAE with H1N1 or H3N2 IAV strains does not trigger MUC1 shedding but instead stimulates an increase in cell-associated MUC1 protein. We observed a similar increase after type I or III interferon (IFN) stimulation; however, inhibition of IFN signaling during H1N1 infection only partially abrogated this increase, indicating that multiple soluble factors contribute to MUC1 upregulation during the antiviral response. In addition to HAE, primary human monocyte-derived macrophages also upregulated MUC1 protein in response to IFN treatment and conditioned media from IAV-infected HAE. Then, to determine the impact of MUC1 on IAV pathogenesis, we developed HAE genetically depleted of MUC1 and found that MUC1 knockout cultures exhibited enhanced viral growth compared to control cultures for several IAV strains. Together, our data support a model whereby MUC1 inhibits productive uptake of IAV in HAE. Infection then stimulates MUC1 expression on multiple cell types through IFN-dependent and -independent mechanisms that further impact infection dynamics. IMPORTANCE Influenza A virus (IAV) targets airway epithelial cells for infection. Large, heavily glycosylated molecules known as tethered mucins extend from the airway epithelial cell surface and may physically restrict pathogen access to underlying cells. Additionally, tethered mucin 1 (MUC1) can be differentially phosphorylated based on external stimuli and can influence inflammation. Given MUC1's multifunctional capability, we sought to define its role during IAV infection. Here, we demonstrate that IAV directly interacts with MUC1 in a physiologically relevant model of human airway epithelium (HAE) and find that MUC1 protein expression is elevated throughout the epithelium and in primary human monocyte-derived macrophages in response to antiviral signals produced during infection. Using CRISPR/Cas9-modified HAE, we demonstrated more efficient IAV infection when MUC1 is genetically ablated. Our data suggest that MUC1 physically restricts IAV uptake and represents a dynamic component of the host response that acts to inhibit viral spread, yielding new insight into mucin-mediated antiviral defense.

Immunofluorescence-Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures.

A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard-to-grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well-differentiated human airway epithelial cell cultures at air-liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses-specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-and provide a protocol for the detection of double-stranded RNA or viral antigen-positive cells by immunofluorescence microscopy. These techniques, together with a post-imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human airway epithelial cultures at air-liquid interface (HAE-ALI) Basic Protocol 2: Viral inoculation of HAE-ALI Basic Protocol 3: Immunofluorescence (IF)-based detection of infected cells in HAE-ALI.

Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality-aspects of infection that may contribute to pathogenesis in vivo.

Human coronavirus alone or in co-infection with rhinovirus C is a risk factor for severe respiratory disease and admission to the pediatric intensive care unit: A one-year study in Southeast Brazil.

OBJECTIVE: We aimed to assess the profile of respiratory viruses in young children hospitalized for acute lower respiratory tract infection (ALRI) and its association with disease severity, defined as need for pediatric intensive care unit (PICU) admission. DESIGN: Prospective observational cohort study. SETTING: A tertiary-care university hospital in Brazil. PATIENTS: Children younger than three years attending the pediatric emergency room with ALRI who were admitted to the hospital. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Nasopharyngeal aspirates were collected from patients from June 1st, 2008 to May 31st, 2009within the first 48 hours of hospitalization. Nasopharyngeal aspirates were tested for 17humanrespiratory viruses by molecular and immunofluorescence based assays. Simple and multiple log-binomial regression models were constructed to assess associations of virus type with a need for PICU admission. Age, prematurity, the presence of an underlying disease and congenital heart disease were covariates. Nasopharyngeal aspirates were positive for at least one virus in 236 patients. Rhinoviruses were detected in 85.6% of samples, with a preponderance of rhinovirus C (RV-C) (61.9%). Respiratory syncytial virus was detected in 59.8% and human coronavirus (HCoV) in 11% of the samples. Co-detections of two to five viruses were found in 78% of the patients. The detection of HCoV alone (adjusted relative risk (RR) 2.18; 95% CI 1.15-4.15) or in co-infection with RV-C (adjusted RR 2.37; 95% CI 1.23-4.58) was independently associated with PICU admission. CONCLUSIONS: The detection of HCoV alone or in co-infection with RV-C was independently associated with PICU admission in young children hospitalized for ALRI.