Avian influenza A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells

Departments of Microbiology and Biology, and Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, New Territories, Hong Kong Special Administration Region, People's Republic of China

^* To whom correspondence should be addressed. Email: paulkschan{at}cuhk.edu.hk.

	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, it was cloned and expressed in human airway epithelial cells (NCI-H292). The apoptotic events at post-transfection were examined by the terminal transferase dUTP nick end labeling (TUNEL) assay, flow cytometric measurement of propidium iodide (PI), annexin V staining, and Western blot analyses with antibodies specific for pro-apoptotic and anti-apoptotic 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 (PARP) and activation of caspases-3, -7, -8 during the NS1-induced apoptosis. 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 (time to >30% apoptosis was reduced from 24 hr to 6 hr post-transfection). Furthermore, after 24 hr post-transfection, there was about a 5.6-fold increase in Fas ligand mRNA expression detected in H5N1-NS1 transfected cells. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A 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.