Ciliated cells promote high infectious potential of influenza A virus through the efficient intracellular activation of hemagglutinin

Influenza viruses utilize host proteases to activate the viral fusion protein, hemagglutinin (HA), into its fusion-competent form. Although proteolytic activation of HA is essential for virus replication, the cell-type dependence of HA activation within the airway epithelium and the subcellular location(s) in which it occurs are not well established. To address these questions, we investigated the proteolytic activation of HA in differentiated human airway epithelial cells using contemporary and historical H1N1 and H3N2 strains. We find that activation is efficient across viral strains and subtypes but depends on cellular tropism, with ciliated cells activating HA more effectively than non-ciliated cells. Similar to prior observations in immortalized cell lines, we find that HA activation occurs intracellularly, constraining the antiviral activity of host-directed protease inhibitors. These results establish that HA activation within the airway epithelium depends on cellular tropism and identify important considerations for the development of protease inhibitors as antivirals.IMPORTANCEInfluenza entry requires the proteolytic activation of the viral fusion protein, HA. Activation occurs as new viruses are produced by infected cells. Efficient proteolytic activation is critical for viral pathogenesis, and inhibiting the requisite proteases could provide an effective host-directed antiviral strategy. To understand cellular constraints on HA activation and its sensitivity to inhibitors, we use complementary approaches to investigate these processes in differentiated airway epithelial cells. We find that ciliated cells activate HA with higher efficiency than non-ciliated cell types, establishing a new mechanism through which cellular tropism and virus infectious potential are connected. We also establish that HA activation begins in the Golgi, which may contribute to the limited efficacy of inhibitors we observe despite their high <i>in vitro</i> potency in a cell-free context.