Study On PB1 Protein Of H7N9 Influenza A Virus Inhibiting The IFN Signaling Pathway By Targeting MAVS

The H7N9 A influenza virus(IAV)was first isolated from people in 2013.As time goes on,the cases of human infection with H7N9 IAV were increasing.Until April 2017,grand total of 1344 people have infected H7N9 IAV,among them have died 511 people,mortality up to 38%,have serious menace to human public health security.At the same time,China poultry turned up high pathogenicity H7N9 IAV,and virus have spread from east coast to Gansu and Xizang in west,give serious lash to poultry trade and live-bird markets.However,H7N9 IAV has infected people or animal pathogenicity and immunologic mechanism still in early stage of research,although research shows IAV can code various interferon antagonist protein,inhibition activate host immune correlation factor,escape host immune reaction,but H7N9 IAV code protein to effect host natural immune signal pathway is not crystal clear.In this study,the cDNA of A/Environment/Suzhou/SZ19/2014(H7N9)strain,isolated from the environment,was used as a template to amplify genes of H7N9 IAV.The resultant genes was cloned into the pRK eukaryotic expression vector.However,to exclude the influence of PB1-F2 that is encoded by PB1 via a frame-shifting manner,two nucleotides at the positions of 96 and 129 in PB1 gene were silently mutated by site-directed mutagenesis,respectively.Dual fluorescent reporting gene system and quantitative real-time PCR(qPCR)were used to screen the related proteins regulating interferon(IFN)signaling pathway;Subsequently,the effects of PB1 protein on the IFN-?,IFN stimulated response element(ISRE)or nuclear regulator factor kappa-B(NF-?B)promoters activity,activated by retinoic-acidinducible gene I CARD(RIG-IN)or myeloid differentiation factor 88(MyD88),were detected.Then,PB1 protein was overexpressed in HEK 293 T cells,and the transcriptional level of IFN-? and its downstream genes were detected by qPCR;the phosphorylation level of TANK-binding kinase 1(TBK1),interforn regulatory factor(IRF3)and inhibitor of NF-?B?(I?B?)was detected by Western blotting;the secretion of antiviral factors from cells was measured by VSV-GFP assay.Further,we determined the target of PB1 through co-immunoprecipitation assay and GST-pull down assay.PB1 was co-transfected dose-dependently with mitochondrial antiviral signaling(MAVS),RIG-I,TNFR-associated factor 3(TRAF3),TBK1,IKK? or IRF3 into HEK 293 T cells to explore the effect of PB1 protein on the expression of these signal molecules by immunoblot.The results showed that H7N9 IAV PB2?PB1 and non-structural protein 1(NS1)can significant inhibition RIG-?N activation IFN-? and ISRE promoter activation,and significant downregulation IFN-? transcriptional level;H7N9 IAV PB1 protein extremely significant inhibition RIG-?N activate IFN-??ISRE and NF-?B promoter activation,but MyD88 activate NF-?B promoter activation has no effect;PB1 protein extremely significant downregulation IFN-? and interferon stimulated gene 56(ISG56)?ISG15 and C-X-C motif chemokine 10 gene(CXCL-10)transcriptional level,induce TBK1?IRF3 and I?B? phosphorylation,inhibition HEK 293 T cell secretion antiviral agent;PB1 protein specifically interacted with MAVS in RLRs signaling pathway,consistent with the results from GST-pull down test.The increased expression of PB1 inhibited MAVS expression,but not RIG-?,TRAF3,TBK1 or IRF3.The preliminary data in the present study demonstrated that PB2,PB1 and NS1 proteins of H7N9 IAV are interferon antagonistic proteins?Those date mentioned above also elucidated that H7N9 PB1 is specifically associated with the important adaptor,MAVS in the the RIG-?like receptor(RLRs)signaling pathway and decreases the MAVS expression,leading to the inhibition of IFNs expression and the blockade of the downstream signaling transduction.