Screening And Function Verification Of Antiviral Genes Against Influenza Virus And HSP27 Negatively Regulates Interferon ? Signaling

The pig is regarded as the mixer of influenza virus gene reassortant,as well as the incubator of new influenza virus strains,so the pig is an important intermediate host involved in transmission of influenza virus.Therefore,there are important public health significances for effective prevention and control of swine influenza virus(SIV)in pigs.Swine flu is a major disease of swine immune suppression,which could bring great harms to pig industry with synergistically other pathogen infection or secondary infection.The innate immune system is the nonspecific defense barrier of body against invading pathogens.The outcome of infection depends on the interaction betwwen incidence of infection and the host defence function.The pig with the a strengthening defence function will show a natural resistance to disease.Therefore,from a long-term point of view,it is at the root of the problem of screening and breeding of antiviral animal against influenza virus using genetic methods to improve resistance to SIV.At present,a main problem is the lack of effective candidate gene for antiviral breeding.Meanwhile,in order to infect the host succesefully,influenza virus adopts various strategies to antagonize host antiviral defense response,especially interferon response.Heat shock protein 27(HSP27)is a widely expressed in cells belonging to small heat.shock proteins.Most viruses can induce high expression of HSP27,but the role of HSP27 in viral infection is still unclear.Previous studies have indicated that HSP27 can participate in the regulation of cell signal transduction pathways,including NF-KB-mediated inflammatory response,but the role of HSP27 in the host IFN signaling is unclear.Therefore,in this study' we firstly used pig transcriptome microarray and bioinformatics platform for the screening candidate antiviral genes against SIV,cloned the genes from the lung tissue or cell,and determined the antiviral effect of them on influenza virus replication through gene overexpression and silencing.We also explored the antiviral mechanism of significant antiviral genes.Besides,we explored the effect of HSP27 protein on the proliferation of the virus and on innate immune signal pathway,to determine the role of HSP27 in virus infection and innate immune signaling pathway.The main results are as follows:1 Analysis of lung tissue transcriptome microarray and Bioinformatics268 differentially expressed(DE)genes were found with clear functional annotation on PID 3 and 214 genes on PID 7.The GO classification of DE genes,showed that onPID 3,the DE genes were involved in many vital functional classes,mainly including signal transduction,immune response,inflammatory response,cell adhesion and cell-cell signalling.Noticeably,the genes associated with immune and inflammatory response showed highly overexpressed.However,on PID 7,the predominant function classes of DE genes included signal transduction,metabolism,transcription,development and transport.Pathway analysis showed that the significant pathways mainly concerned with Cell adhesion molecules,Cytokine-cytokine receptor interaction,Toll-like receptor signaling pathway and MAPK signaling pathway,suggesting that the host took different strategies to activate these pathways so as to prevent virus infections at the early stage.Furthermore,the most significant pathways on PID 7 switched to PPAR signaling pathway and complement and coagulation cascades,showing that the host might start to repair excessive tissue damage by anti-inflammatory functions.2 Gene cloning,expression and antiviral effect on influenza virusIn order to verify the antiviral effects of interferon signaling pathway molecules and IFN-stimulated genes on the proliferation of influenza virus,35 genes were firstly cloned from poly(I:C)-treated PAM cell.Overexpression experiments showed that interferon signaling adaptors,such as TBK1 and IRFs,inhibited obviously virus proliferation;interferon induced genes,such as IFITs and GBPs,also has significant antiviral effect,but less than interferon signaling adaptors.In addition,we also found that the pig host defence peptides PAMP23 and PG4 can significantly inhibit the replication of the avian influenza virus,with dose-dependent effect.Moreover,IRF1 has the most significant antiviral effect,which could reduce the virus titer of 100 times,even better than that of IRF3.and IRF1 significantly enhance the activities of VISA-induced IFN-?-luc,ISRE-luc and NF-?b-luc.3 Antiviral activity of porcine GBP1GBP1 is an important gene regulated by IRF1,and also posseses very significant antiviral activity against varied subtypes of influenza virus.And GBP1 had significant inhibitory effect on three forms of RNA of influenza virus.After GBP1 silencing,virus proliferation in PK-15 cells was significantly increased,which further proved that GBP1 is an antiviral protein.Antiviral function of porcine GBP1 depends on in its N terminal GTPase domain,and the 47 and 48 amino acids in GTP binding sites in N terminal.GBP1 also reduced polymerase activity of influenza virus significantly.Co-IP showed that GBP1 could interact with NP and NS1 protein of influenza virus,and interaction strength with NP protein was significantly greater than that with the NS1.Laser scanning confocal microscope displayed GBP1 and NP were colocalized around the nucleus.4 Effect of HSP27 on the proliferation of influenza virusOverexpression of HSP27 significantly promoted the proliferation of the H5N1 virus in the infected cells.Especially at 36h,the virus titer in the overexpression of HSP27 is about 10 times of the control cells for virus titer.Meanwhile,overexpression of HSP27 can promote the proliferation of VSV virus,indicating that HSP27 is an unspecific factor for promoting virus replication.On the contrary,knockout of HSP27 decreased significantly the virus titer.Thus,HSP27 is an essential host factor in viral replication cycle.5 Effect of HSP27 on IFN signaling pathwayPromoter reporter assays showed that overexpression of HSP27 significantly inhibited the Sendai virus and RIG-1,MDA5,VISA,TBK1 and IKKi induced interferon promoter activity,and reduced the level of IRF3 phosphorylation induced by VISA and TRIF protein.In contrast,silencing of HSP27 enhanced the interferon promoter activity.At the same time,it was also shown that HSP27 can negatively regulate type I interferon signaling transduction by IRF3 and NF-?B signaling.In addition,HSP27 was found distribution at mitochondria during the course of virus infection.6 The mechanism of HSP27 on the regulation of IFN signalingCo-immunoprecipitation experiments showed that HSP27 interacted with VISA,TRAF3 and TRAF6,leading to the degradation of TRAF3 and TRAF6 protein.Ubiquitination experiment found that HSP27 can cause the degradation of TRAF3 and TRAF6 proteins through the K48-ubiquitination pathway,which inhibit the production of interferon mediated by IRF3 and NF-?B.Besides,negative regulation of HSP27 in the IFN depends on the 82 phosphorylation.Our findings suggest that HSP27 functions as a negative regulator of innate IFN response by preventing TRAF3 from forming protein complexes with critical downstream transducers and effectors.