Mechanisms Of H5N1 Virus-induced Autophagy

Influenza A Virus(IAV)is one of the most common pathogens that causes respiratory infection.Avian influenza virus subtypes such as H5N1 and H9N2 mainly infects birds and can be divided into highly pathogenic and low pathogenic IAV.Avian influenza not only causes great economic losses to poultry industry,but also poses a great threat to human health..Better understanding of the pathogenesis of avian influenza will help design novel strategy to control IAV infection.Autophagy is an intrinsic regulation mechanism for organisms to adapt to different environments.Autophagy can help the cell to clear intracellular pathogens or can be used by pathogens to escape the host's immune response.Autophagy is regulated by many signaling pathways,including MAPK-JNK pathway,AMPK pathway and mTOR pathway.C-Jun terminal kinase(JNK)facilitates autophagy by phosphorylating Bcl-2 and disrupting its interaction with Beclin-1.AMPK activation induces autophagy by inhibiting the activity of mTOR and phosphorylating the Ser555 and Ser317 site of ULK1.Whether these signaling pathways are involved in regulating IAV-induced autophagy remains unclear.In the present study,we employed 293T cells to study the molecular mechanism of avian influenza virus-induced autophagy and its effect on virus replication.Different IAV subtypes can differentially activate JNK,H5N1 but not H1N1 virus activates JNK.However,the mechanism of IAV-induced JNK activation is still unclear.As a member of the MAPK family,JNK is regulated by a variety of upstream proteins,such as TAK1,ASK1,MEKK1,etc.TAK1 is an important serine/threonine kinase that regulates cell survival,differentiation,apoptosis and immune response.TAK1 is involved in activating several important kinases in cells,including p38,JNK and IKK.The main purpose of this study is to investigate whether H5N1 virus can activate downstream MKK4 via TAK1 signaling pathway,thereby promoting JNK activation and autophagy.We first analyzed the expression of autophagy-related proteins in IAV-infected 293T cells s by Western blotting.We found that both H5N1 and H1N1 viruses increased LC3 lipidation.However,H5N1 virus inhibited the expression of p62,while H1N1 viru did not affect the expression ofp62.Using Vero cells stably transfected with GFP-RFP-LC3,we found that H5N1 virus induced formation of autolysosomes.H5N1 virus had no effect on AMPK signaling pathway,but activated JNK signaling pathway.To further explore how H5N1 virus activate JNK signaling pathway,we used TAK1 inhibitor 5Z,ASK1 inhibitor MSC and TAK1 knockout to study which kinase regulates JNK upstream.We found that H5N1 virus activated JNK signaling pathway through TAK1 rather than ASK1.The NS 1 protein of H5N1 but not HINI and H9N2 viruses was responsible for JNK and MKK4 protein and its downstream molecule Bcl-2.These results suggest that H5N1 virus may promote Bcl-2 phosphorylation,induce the dissociation of Bcl-2-Beclin1 complex through the activation of Bcl-2,and ultimately promote cell autophagy.We further studied whether TAK1 is responsible for H5N1 virus-induced autophagy and impacts viral replication.We found that 5Z significantly inhibited autophagy in H5N1 virus-infected-293T cells and the synthesis of viral protein,lowerd virus titer.These results suggest that TAK1-mediated autophagy can promote the replication of H5N1 virus.In conclusion,our study demonstrated that H5N1 virus induced complete autophagy,while H1N1 virus induced incomplete autophagy.Secondly,we demonstrated that H5N1 virus activates JNK through the TAKI-MKK4 pathway.Finally,we showed that autophagy could promote the replication of H5N1 virus in 293T cells.H5N1 virus had no effect on AMPK activity,suggesting that H5N1 virus induces autophagy in an AMPK-independent manner.