Role Of C-jun Terminal Kinase(JNK)Activation In Influenza A Virus-induced Autophagy And Replication

Influenza A virus(IAV)infects a wide range of avian and mammalian hosts,whereas influenza B virus infects only humans.IAV can be further classified into various subtypes based on the combination of two viral surface glycoproteins,e.g.,hemagglutinin(HA)and neuraminidase(NA).A total of 17 HA and 10 NA subtypes have been identified so far.Among them,several reassortant IAV genotypes,including H5N1,H9N2,H7N2,and H6N5,cause sporadic fatal infections in humans.Autophagy is a catabolic process that mediates the degradation of damaged organelles,the unneeded proteins,and the invading microbes.More autophagy-related proteins involved in the formation of autophagy have been found.Autophagy is initiated by the activation of VPS34,a Class III PI-3 kinase in the preinitiation complex,which contains several other proteins,including Beclin-1,ATG14L,and VSP15.VPS34 is activated by UNC-51-like kinase 1(ULK1),a serine/threonine kinase that interacts with ATG13 and FIP200.Once activated,this pre-initiation complex catalyzes phosphatidylinositol(PI)-4,5 to PI 3-phosphate(PI3P),a phospholipid involved in the elongation and nucleation of the double membrane to form autophagosomes.ULK1 activity is regulated by mTOR and AMPK,These two kinases phosphorylate ULK1 at different serine residues and have the opposite effect on ULK activity:mTOR phosphorylates ULK1 at serine 757(ULK1S757)and inhibits its activity;whereas AMPK phosphorylates ULK1 at multiple sites,including the serine residues 317,555,and 777(ULK1S317/S555/S717),and activates it.c-Jun terminal kinase(JNK),a stress-activated protein kinase,regulates autophagy by phosphorylating Bcl-2 and freeing its binding from Beclin-1.Beclin-1 becomes available for the formation of the preinitiation complex.Alternatively,Beclin-1 can be phosphorylated by AMPK and freed from the Bcl-2-Beclim-1 complexThe non-structural protein 1(NS1)of different influenza A virus(IAV)strains can differentially regulate the activity of the c-Jun terminal kinase(JNK)and PI-3 kinase(PI3K).Whether differential JNK and PI3K activation impacts autophagy and IAV replication differently remains uncertain.Our present study aims to determine the effect of reciprocal JNK and PI3K activation by three different IAV subtypes on autophagy and virus replication in chicken embryonic fibroblast cells(CEF).Here we report that H5N1(A/mallard/Huadong/S/2005)influenza A virus induced functional autophagy,as evidenced by increased levels of LC3 lipidation and decreased p62 levels in a time-and dose-dependent manner.H5N1 virus-induced autophagy was further confirmed by the ability of bafilomycin and chloroquine,two inhibitors of autophagy flux,to increase LC3-II levels but block p62 degradation.Confocal microscopy revealed that H5N1 induced the formation of the red puncta of autolysosomes in LC3-GFP(green fluorescent protein)-RFP(red fluorescent protein)lentivirus-infected DF-1 cells.H9N2(A/chicken/Shanghai/F/98)virus modestly induced autophagy,whereas H1N1 virus(A/PR/8/34,PR8)did not induce but rather blocked autophagic flux,as evidenced by increased p62 expression.H5N1 virus activated JNK but inhibited the PI-3 kinase pathway,as evidenced by increased JNK phosphorylation but decreased phosphorylation of AKTS473,S6K1T389,S6S235/236,and ULK1S757.In contrast,N9N2 virus led to modest JNK activation and strong PI-3 kinase activation;whereas H1N1 virus activated the PI-3 kinase pathway but did not activate JNK.SP600125,a JNK inhibitor,inhibited H5N1 virus-induced autophagy and virus replication in CEF.Our study provides unambiguous evidence that H5N1 virus induces complete autophagy,whereas H1N1 virus blocks the autophagic flux.Our study suggests that JNK activation benefits H5N1 virus replication by inducing autophagy.