title: Avian influenza virus A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells.
abstract: Avian H5N1 influenza virus causes a remarkably severe disease in humans, with an overall case fatality rate of greater than 50%. Human influenza A viruses induce apoptosis in infected cells, which can lead to organ dysfunction. To verify the role of H5N1-encoded NS1 in inducing apoptosis, the NS1 gene was cloned and expressed in human airway epithelial cells (NCI-H292 cells). The apoptotic events posttransfection were examined by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay, flow cytometric measurement of propidium iodide, annexin V staining, and Western blot analyses with antibodies specific for proapoptotic and antiapoptotic proteins. We demonstrated that the expression of H5N1 NS1 protein in NCI-H292 cells was sufficient to induce apoptotic cell death. Western blot analyses also showed that there was prominent cleavage of poly(ADP-ribose) polymerase and activation of caspase-3, caspase-7, and caspase-8 during the NS1-induced apoptosis. The results of caspase inhibitor assays further confirmed the involvement of caspase-dependent pathways in the NS1-induced apoptosis. Interestingly, the ability of H5N1 NS1 protein to induce apoptosis was much enhanced in cells pretreated with Fas ligand (the time posttransfection required to reach >30% apoptosis was reduced from 24 to 6 h). Furthermore, 24 h posttransfection, an increase in Fas ligand mRNA expression of about 5.6-fold was detected in cells transfected with H5N1 NS1. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A virus H5N1 induced apoptosis in human lung epithelial cells, mainly via the caspase-dependent pathway, which encourages further investigation into the potential for the NS1 protein to be a novel therapeutic target.
authors.affiliation: Department of Microbiology, 1/F Clinical Science Building, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, People's Republic of China.
authors.forename: W Y
authors.initials: WY
authors.lastname: Lam
authors.forename: Julian W
authors.initials: JW
authors.lastname: Tang
authors.forename: Apple C M
authors.initials: AC
authors.lastname: Yeung
authors.forename: Lawrence C M
authors.initials: LC
authors.lastname: Chiu
authors.forename: Joseph J Y
authors.initials: JJ
authors.lastname: Sung
authors.forename: Paul K S
authors.initials: PK
authors.lastname: Chan
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D015153:Blotting, Western; D001980:Bronchi / Q000166:cytology / Q000201:enzymology / Q000821:virology*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology / Q000821:virology; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D053124:Influenza A Virus, H5N1 Subtype / Q000378:metabolism* / Q000472:pathogenicity; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014774:Virulence
chemical_list: D017931:DNA Primers; C108309:NS protein, influenza virus; D017361:Viral Nonstructural Proteins; D020169:Caspases