PB1-F2 Of Influenza A Virus Interacts With P58^IPK And Regulates PKR Signaling

Influenza virus（IV）was the single-stranded negative chain RNA virus which could cause people,dogs,horses,pigs and poultry suffering from influenza disease.The influenza A virus（IAV）was the strongest pathogenicity in type A,B and C three influenza viruses and it often causes outbreaks or epidemic of influenza,it was the main pathogen of human respiratory infectious epidemic disease and livestock death.protein was one of the key protein of IAV,its existence and expression level had a close contact with the fatality rate of IAV,which was found in several outbreaks of influenza pandemic.PB1-F2 had many functions,it had been discovered that it could promote the proliferation of viral replication,locate on the mitochondria of host cell to mediate the apoptosis of mitochondrial pathways,induce inflammatory response and improve the viral polymerase activity.Though the structure and function of PB1-F2 protein had been researched a lot at home and abroad,the mechanism and impact on the host cell remained to be further exploration.p58^IPK was the intracellular inhibitor of protein kinase R（PKR）which was induced by interferon and activated by double-stranded RNA.When the influenza virus infected host cells,p58 IPK was activated and then suppressed the phosphorylation of protein kinase R or e IF2α,inhibiting signaling transduction pathways,reducing the apoptosis and inflammation of host cells,promoting protein expression and replication of virus.The interaction between PB1-F2 and p58 IPK was confirmed by yeast hybrid experiment.To further demonstrate in viro,we built the p GEX4T-1-PB1-F2 recombinant plasmid through the molecular cloning technology and certified the interaction through GST pull-down.Then PB1-F2 interacted with p58 IPK in vivo was proved through Co-immunoprecipitation（Co-IP）.Last,PB1-F2 interacted with p58 IPK and they colocated in the cytoplasm were attested by immunofluorescence experiment.Arter PB1-F2 protein was overexpressed,the m RNA transcription and protein expression of p58 IPK had been up-regulated comparing with the control group through Real-time PCR and Western blot experiment.The above experiments illustrated the interaction between PB1-F2 and p58 IPK,the m RNA transcription and protein expression of p58 IPK were up-regulated by PB1-F2 protein.While p58 IPK was the the intracellular inhibitor of PKR and restrained the PKR signaling pathways,then PKR maid be regulated by PB1-F2.Above interference once confirmed,it would provide a new basis for PB1-F2 regulated PKR signaling pathway.Though PKR was inactive in host cells under normal circumstance,it could participate in a series of signaling transduction and information transmission（such as NF-κB signaling pathways and STAT signaling pathways and so on）once it was activated into phosphorylated PKR（p-PKR）.The p-PKR could asserted those signaling pathways to regulate the secretion of interferon,viral replication,inflammation response and apoptosis of cells.PB1-F2 and PKR were associated with viral replication and inflammatory response,so we carried a large amount of research on PB1-F2 regulated PKR signaling pathways and PKR influenced viral replication.Arter PB1-F2 was overexpressed,the m RNA transcription level of PKR and its downstream factor IL-6,the protein phosphorylation level of PKR and its downstream protein e IF2α and stat3 were all down-regulated comparing with the control group through the Real-time PCR and Western blot experiment,and the two trends were dose dependent.The red fluorescent Red-PKR protein was inhibited after GFP-PB1-F2 was overexpressed through immunofluorescence experiment,which further proved the PB1-F2 suppressed the protein expression of PKR.The m RNA transcription of IL-6 and stat3,the protein espression of e IF2α and stat3 were all prominent down-regulated after using si RNA to interfer PKR gene expression.After PB1-F2 was overexpressed,the transcription activity of NF-κB-luc reporter was degraded,while after using si-PKR,the trend of transcription activity was more obvious.In addition,the m RNA transcription and protein expression of NP and M protein of influenza virus were down-regulated after PKR protein was overexpressed and the influenza virus PR8 infected human lung cancer cells through Real-time PCR and western blot experiment.But the m RNA transcription and protein expression of NP and M protein was significantly up-regulated comparing with control group after si-PKR interfered the gene expression of PKR,and the trend was dose dependent.To sum up,PB1-F2 protein of IAV interacted with p58 IPK and up-regulated the m RNA transcription and protein expression to suppress the PKR signaling pathways and then promoted viral replication.This experiment could add more experimental data for the research of influenza virus pathogenic mechanism and provide a new direction for the prevention and control of influenza virus.