Regulation Of Autophagy By Influenza Virus And Its M2 Ion Channel Protein

Influenza viruses are globally important human respiratory pathogens. They cause seasonal epidemics and occasional worldwide pandemics. In March 2013, a novel avian-origin influenza virus subtype (A/H7N9) emerged in eastern China causing global concerns about its pandemic potential. The outstanding feature of influenza virus is their capacity for regulating host cell conservative pathways. But the viral factors and the underlying molecular details of this virus-host interplay are poorly understood.Autophagy is an intracellular degradation process in which cytoplasmic materials, including long-lived proteins and damaged organelles, are directed to the lysosome by a membrane-mediated process. The hallmark of autophagy is the emergence of double membrane vesicles called autophagosomes. Autophagy affects a multitude of health and disease states including development, cancer and infectious diseases. Recent studies reported that autophagy was involved in influenza virus infection. Influenza virus was shown to enhance autophagosome accumulation in vitro and in vivo. But how influenza virus affects autophagy and the underlying mechanisms are still controversial.To test if autophagy can be affected by influenza virus infection, we infected cells with the influenza A/Hong Kong/8/68(H3N2) or A/WS/33(H1N1) and investigated the autophagy changes in infected cells. Both strains of influenza virus induced the autophagosome marker LC3-II accumulation in infected cells. Also, the viruses induced punctuate GFP-LC3 accumulation in infected cells. Notably, in the two strains of influenza virus used, influenza A/Hong Kong/8/68(H3N2) is amantadine sensitive whereas influenza A/WS/33(H1N1) is amantadine resistant. Strikingly, after influenza virus M2 ion channel protein inhibitor amantadine treatment, no apparent autopahgosomes accumulation in amantadine-sensitive virus infected cells but still prominent in amantadine-resistant virus infected cells. The influenza virus neuraminidase inhibitor oseltamivir, did not affect autophagosomes accumulation in virus infected cells. These results suggested that influenza virus induced autophagosomes accumulation with its M2 protein ion channel activity dependent manner.To further investigate whether influenza virus M2 protein ion channel activity sufficiently induced autophagosomes accumulation we used the M2 proteins of H5Nland the newly-outbreak H7N9. Notably, the S31N mutation causes M2 ion channel protein resistant to amantadine. Expression of the amantadine sensitive H5N1-M2 (WT) or amantadine resistant mutant H5N1-M2 (S31N) could induce remarkable autophagosomes accumulation. Amantadine inhibited autophagosomes accumulation induced by H5N1-M2 (WT) but not H5N1-M2 (S31N) mutant. Also, expression of H7N9-M2 or its amantadine sensitive mutant H7N9-M2 (N31S) could induce remarkable autophagosomes accumulation. Remarkably, amantadine could only inhibit autophagosomes accumulation induced by H7N9-M2 (N31S) but not H7N9-M2. Taken together, these results indicated that influenza virus M2 protein sufficiently induced autophagosomes accumulation with its ion channel activity dependent manner.Cellular autophagy degrades bulk proteins and target contents through autophagosomes formation and fusion with lysosomes known as autolysosomes. Surprisingly, autophagosomes induced by M2 proteins failed to fuse with lysosomes as indicated by the confocal microscopy analysis of GFP-LC3 dots with lysosome-associated membrane protein 2 (LAMP2). To further confirm this observation, we analyzed the turnover of the autophagy substrate protein p62. M2 proteins induced p62 accumulation on autophagosomes and inhibited p62 degradation with M2 ion channel activity dependent manner. Accordingly, influenza virus infection also induced insufficient fusion of autophagosomes with lysosomes and inhibited the autophagic degradation of p62. Collectively, these results suggested that influenza virus M2 protein arrested autophagy by blocking the autophagosome-lysosome fusion and inhibiting autophagic degradation.Influenza virus M2 is a proton ion channel protein expressing on intracellular membrane system (endoplasmic reticulum (ER) or trans-Golgi network (TGN)) and neutralizing the pH of these intracellular compartments. We hypothesized that M2 might also act on autophagosomes, because the membrane component of autophagosomes mainly derived from the endoplasmic reticulum (ER) or trans-Golgi network (TGN). We found that M2 protein indeed localized on autophagosomes. So we proposed that influenza virus M2 might block the autophagosomes fusion with lysosomes by disrupting the pH gradient of autophagosomes.We explored the previously unappreciated function of ion channel activity of M2 protein during influenza virus induced autophagosomes accumulation. Strikingly, we found that influenza virus M2 ion protein was an important viral factor to arrest autophagy by blocking the autophagosome-lysosome fusion and inhibiting the autophagic degradation. Taken together, we described the elaborate regulation of autophagy by influenza virus M2 protein. This would expand our knowledge of the influenza-host interaction and may provide clues for the prevention of the potential H7N9 pandemic.