A number of studies have demonstrated that influenza virus infection can induce apoptosis in a variety of cell lines, but the mechanism of this effect remains to be characterized [
1‐
12]. Previous studies thought that apoptosis is a host defense response that limits the virus replication [
29], but recent evidence showed that the induction of apoptosis is essential for virus mRNA synthesis and propagation [
16,
30,
31]. Viral proteins from different strains of human influenza viruses have been reported to have proapoptotic or antiapoptotic functions in human cells [
7,
14‐
18]. The importance of non-structural protein NS1 in the viral pathogenesis, especially its role in the virus-induced apoptosis, has been recently underlined [
8,
17,
23,
25,
27]. A highly controversial question is whether this multifunctional protein is a proapoptotic or antiapoptotic factor in infected cells. Schultz-Cherry
et al. reported that the expression of NS1 protein of H5N9 was sufficient to induce apoptosis in MDCK and Hela cells [
8]; Lam
et al demonstrated recently that H5N1 NS1 protein could induce apoptosis in human airway epithelial cells (NCI-H292) [
26]. It was also reported that a poor-apoptosis-inducer strain could be converted into a strong-inducer strain by the NS1 gene substitution by reverse genetics, and vice versa [
25]. Discordantly, the NS1 protein has been also shown to inhibit apoptosis. For examples, Zhirnov
et al. found that H1N1 NS1 protein had IFN-dependent antiapoptotic potential and down-regulated the apoptotic response in virus-infected in cultured cells and chicken embryos [
18]; Ehrhardt
et al found that the NS1 proteins of H1N1 and H7N7 activated the phosphatidylinositol 3-kinase (PI3K/Akt) pathway to mediate antiapoptotic signaling responses [
27]. The discrepancy has confused our understanding to the role of NS1 protein in influenza virus-induced apoptosis, highlighting that further characterization is needed to exclude the possibility resulted from the different cell lines and virus strains in each experiment [
20]. In the present study, we cloned the NS1 gene from the influenza A/chicken/Jilin/2003 virus (H5N1) and expressed the NS1 protein in the human alveolar basal epithelial cell A549. With electron microscopic and flow cytometric analyses we verified that the expression of H5N1 NS1 protein was capable to induce apoptotic events posttransfection in the A549 cells. The biological basis accounting for the severity of H5N1 infection in humans is still unknown. Our current experimental data, together with the apoptotic observations in the alveolar epithelial cells of two patients who died of H5N1 infection [
13], have provided convincing evidence to the critical role of H5N1-encoded NS1 protein in inducing apoptosis.
Apoptosis, or programmed cell death, involves a series of biochemical events that lead the cells undergo characteristic morphological changes, including blebbing, shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation [
32]. Sequential activation of caspases cascade plays a central role in the execution-phase of cell apoptosis. Recently, It has been reported that avian influenza virus A/HK/483/97(H5N1) NS1 protein-induced apoptosis in human lung epithelial cells is mainly via the caspase-dependent pathway [
26], which encourages further investigation into the potential of the NS1 as a novel therapeutic target. To delineate the apoptotic pathway, we measured the expression of caspase-3 and caspase-9 by Western blotting and their enzyme activities by colorimetric Assay. The data demonstrated that these two apoptosis markers were significantly activated in H5N1 NS1-transfected A549 cells, consistent with the previous studies. Therefore, we conclude that the NS1 protein encoded by avian influenza A virus H5N1 can induce apoptosis in human respiratory epithelial cells via the caspase-dependent pathway. Since the apoptotic destruction of host cells has been thought to contribute the severe disease, we can predict that drugs that can prevent this specific process may reduce disease severity and improve clinical outcomes. Therefore, further investigations to clarify whether the NS1 protein and its apoptotic pathway is worthwhile therapeutic targets for treating H5N1 infection in humans should be considered.