title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection.
abstract: We sought to determine the mechanisms by which influenza infection of human epithelial cells decreases cystic fibrosis transmembrane conductance regulator (CFTR) expression and function. We infected human bronchial epithelial (NHBE) cells and murine nasal epithelial (MNE) cells with various strains of influenza A virus. Influenza infection significantly reduced CFTR short circuit currents (Isc) and protein levels at 8 hours postinfection. We then infected CFTR expressing human embryonic kidney (HEK)-293 cells (HEK-293 CFTRwt) with influenza virus encoding a green fluorescent protein (GFP) tag and performed whole-cell and cell-attached patch clamp recordings. Forskolin-stimulated, GlyH-101-sensitive CFTR conductances, and CFTR open probabilities were reduced by 80% in GFP-positive cells; Western blots also showed significant reduction in total and plasma membrane CFTR levels. Knockdown of the influenza matrix protein 2 (M2) with siRNA, or inhibition of its activity by amantadine, prevented the decrease in CFTR expression and function. Lysosome inhibition (bafilomycin-A1), but not proteasome inhibition (lactacystin), prevented the reduction in CFTR levels. Western blots of immunoprecipitated CFTR from influenza-infected cells, treated with BafA1, and probed with antibodies against lysine 63-linked (K-63) or lysine 48-linked (K-48) polyubiquitin chains supported lysosomal targeting. These results highlight CFTR damage, leading to early degradation as an important contributing factor to influenza infection-associated ion transport defects.
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA.
authors: James David
authors: JD
authors: Londino
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA.
authors: Ahmed
authors: A
authors: Lazrak
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA.
authors: James W
authors: JW
authors: Noah
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA.
authors: Saurabh
authors: S
authors: Aggarwal
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA.
authors: Vedrana
authors: V
authors: Bali
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA.
authors: Bradford A
authors: BA
authors: Woodworth
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA.
authors: Zsuzsanna
authors: Z
authors: Bebok
authors: *Department of Anesthesiology, Pulmonary Injury and Repair Center, and Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Southern Research Institute, Birmingham, Alabama, USA; and Department of Surgery, Division of Otolaryngology, School of Medicine, University of Alabama, Birmingham, Alabama, USA sadis@uab.edu.
authors: Sadis
authors: S
authors: Matalon
mesh_terms: D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology / Q000821:virology; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D007251:Influenza, Human / Q000378:metabolism / Q000473:pathology / Q000821:virology; D017136:Ion Transport; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D009336:Necrosis; D018408:Patch-Clamp Techniques; D059748:Proteolysis; D014162:Transfection; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D000086082:Viroporin Proteins
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014763:Viral Matrix Proteins; C505032:CFTR protein, human; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins
keywords: DsiRNA; NHBE cells; amantadine; patch clamp; short circuit current