title: Suppression of influenza A virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin A1.
abstract: 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 A1 (Baf-A1), 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-A1 also have anti-IAV activity, but without toxic effects. Thus we assessed the antiviral activity of Baf-A1 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-A1 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-A1 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-A1 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-A1, 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-A1 is an effective inhibitor of IAV replication, without impacting host cell viability.
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada;
authors.forename: Behzad
authors.initials: B
authors.lastname: Yeganeh
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Saeid
authors.initials: S
authors.lastname: Ghavami
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada;
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Kroeker
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada;
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Mahood
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada;
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Stelmack
authors.affiliation: Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Surgery, University of Manitoba, Winnipeg, Manitoba, Canada; and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Klonisch
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Coombs
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada andrew.halayko@umanitoba.ca.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Halayko
mesh_terms: D056809:Alveolar Epithelial Cells / Q000821:virology*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001343:Autophagy; D045744:Cell Line, Tumor; D004285:Dogs; D004353:Drug Evaluation, Preclinical; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000502:physiology*; D018942:Macrolides / Q000494:pharmacology*; D061985:Madin Darby Canine Kidney Cells; D053585:Virus Attachment; D057074:Virus Release / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D018942:Macrolides; C040929:bafilomycin A1
keywords: apoptotic cell death; autophagy; influenza A virus; low-dose bafilomycin A1; noncytotoxic