Suppression of influenza A virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin A1

Subcellular trafficking within host cells plays a critical role in viral life cycles, including influenza A virus (IAV). Thus targeting relevant subcellular compartments holds promise for effective intervention to control the impact of influenza infection. Bafilomycin A(1) (Baf-A(1)), when used at relative high concentrations (>= 10 nM), inhibits vacuolar ATPase (V-ATPase) and reduces endosome acidification and lysosome number, thus inhibiting IAV replication but promoting host cell cytotoxicity. We tested the hypothesis that much lower doses of Baf-A(1) also have anti-IAV activity, but without toxic effects. Thus we assessed the antiviral activity of Baf-A(1) at different concentrations (0.1-100 nM) in human alveolar epithelial cells (A549) infected with IAV strain A/PR/8/34 virus (H1N1). Infected and mock-infected cells pre- and cotreated with Baf-A(1) were harvested 0-24 h postinfection and analyzed by immunoblotting, immunofluorescence, and confocal and electron microscopy. We found that Baf-A1 had disparate concentration-dependent effects on subcellular organelles and suppressed affected IAV replication. At concentrations >= 10 nM Baf-A(1) inhibited acid lysosome formation, which resulted in greatly reduced IAV replication and release. Notably, at a very low concentration of 0.1 nM that is insufficient to reduce lysosome number, Baf-A(1) retained the capacity to significantly impair IAV nuclear accumulation as well as IAV replication and release. In contrast to the effects of high concentrations of Baf-A(1), very low concentrations did not exhibit cytotoxic effects or induce apoptotic cell death, based on morphological and FACS analyses. In conclusion, our results reveal that low-concentration Baf-A(1) is an effective inhibitor of IAV replication, without impacting host cell viability.