Study On The Mechanism Of PRRSV NSP11 Inhibiting Type ? Interferon Signaling Pathway

Innate immunity is the first line of defense against pathogenic microbial invasion,in which type? interferon(IFN-?/IFN-?)plays an important role in antiviral processes.When the virus infected the host cell,the viral pathogen-associated molecular patterns(PAMPs)can be recognized by the pattern recognition receptor(PRRs),thereby activating the downstream signaling molecule to induce type? IFNs(IFN-?/IFN-?)production.After secretion,type? IFNs bind to a heterodimeric receptor composed of IFNAR1 and IFNAR2 on adjacent cell surfaces to activate intracellular janus kinase 1(JAK1)and tyrosine kinase 2(TYK2).Subsequently,signal transducer and activator of transcription 1(STAT1)and STAT2 are then phosphorylated by JAK1 and TYK2,inducing heterodimer formation of STAT1 and STAT2.The heterodimers and IFN regulatory factor 9(IRF9)further form IFN-stimulated gene factor 3(ISGF3),which is transferred into the nucleus and binds to the interferon-stimulating response element(ISREs)to trigger the transcription and translation of hundreds of interferon-stimulating genes(ISGs).As effective antiviral factors,ISGs help to establish antiviral status for host and resistant to viral infection.Porcine reproductive and respiratory syndrome(PRRS)is an immunosuppressive disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which has caused great economic losses to the global pig industry.Previous studies indicated that PRRSV has evolved a series of mechanisms to evade host immunity.PRRSV nsp1?/1?,nsp2,nsp4,nsp11 and N proteins can inhibit the expression of interferon;PRRSV can also inhibit type? interferon signal transduction,but in addition to nsp1? and N,the action mechanism of other proteins is unclear.PRRSV nsp11 has nidovirus uridylate-specific endoribonuclease(Nendo U)activity,which is important for PRRSV replication.In this study,PRRSV nsp11 was used as the research object to explore its role in the type? interferon signaling pathway and further explained the molecular mechanism.The main contents are as follows:1.PRRSV nsp11 and its endoribonuclease activity mutants inhibit ISRE promoter activation and ISGs expression.To explore whether PRRSV nsp11 has the ability to inhibit IFN-?-induced ISRE promoter activation and expression of ISGs.The eukaryotic expression plasmids of nsp11 and its endoribonuclease activity mutants(H129A,H144 A,K173A)were transfected respectively into HEK-293 T cells,and the double luciferase reporter system and real-time fluorescence quantitative PCR assay results showed that nsp11 and its mutants could inhibit ISRE promoter activation and expression of ISGs,indicating that nsp11 prevents type? interferon signaling independently of Nendo U activity.To further explore the possible mechanism of action,the eukaryotic expression plasmids of nsp11 and its mutants(H129A,H144 A,K173A)were transfected respectively into HEK-293 T cells and stimulated with IFN-? for 6 h.Western blot showed that nsp11 and its mutants(H129A,H144 A,K173A)did not affect the phosphorylation of STAT1 and STAT2,and did not affect the expression of STAT1,STAT2 and IRF9.However,the experiment of nuclear and cytoplasmic extraction in HEK-293 T cells showed that nsp11 and its mutants(H129A,H144 A,K173A)prevented the ISGF3 complex(p-STAT1,p-STAT2 and IRF9)into the nucleus.2.PRRSV nsp11 and its endoribonuclease activity mutants interact with IRF9.STAT1,SATA2,and IRF9 play an important role in the type? interferon signaling pathway,which can form ISFG3 complex into nucleus,and induces the ISGs expression.In order to explore the mechanism how nsp11 inhibits the ISGF3 complex into the nucleus,we examined whether nsp11 interacted with STAT1,STAT2 or IRF9 by immunoprecipitation assay.Nsp11 or mutants(H129A,H144 A and K173A)were co-transfected into HEK-293 T cells with STAT1,STAT2 or IRF9 eukaryotic expression plasmids respectively.Immunoprecipitation results showed that nsp11 and mutants interacted with porcine IRF9.In order to identify the interaction domain of IRF9 with nsp11,four truncated mutants with different regions of IRF9 were constructed.The eukaryotic expression plasmids of nsp11 or H129 A were co-transfected into HEK-293 T cells with different IRF9 truncated mutants.The immunoprecipitation results showed that nsp11 and H129 A bound to the IAD domain of IRF9,and finally the ISGF3 complex was retained in the cytoplasm.3.PRRSV nsp11 and its endoribonuclease activity mutants inhibit ISGF3-induced ISRE activation.Since STAT2 also interacts with the IAD domain of IRF9,nsp11 and STAT2 may competitively bind IRF9.To explore whether nsp11 inhibited the formation of ISGF3 complexes,the eukaryotic expression plasmids of nsp11 or H129 A were co-transfected into HEK-293 T cells along with STAT1,STAT2 and IRF9.After 6 h with IFN-? stimunation,the immunoprecipitation results showed that nsp11 and H129 A impaird the formation of IFN-induced ISGF3 complex.To study biological effects on inhibition of ISGF3 formation by nsp11,the eukaryotic expression plasmids of nsp11 or mutants(H129A,H144 A and K173A)were co-transfected with STAT1,STAT2 and IRF9 into HEK-293 T cells.The luciferase reporter assay results showed that nsp11 and mutants significantly inhibited ISGF3-induced ISRE promoter activation.In summary,PRRSV nsp11 was used as the reseach object to explore its role in antagonizing type? interferon signaling pathway.The results shown that PRRSV nsp11 inhibited ISRE promoter activation and ISGs expression,but independent of its endoribonuclease activity.Further studies shown that PRRSV nsp11 and its mutants interacted with the IAD region of ISGF3 complex important component IRF9,then inhibiting ISGF3-induced ISRE promoter activation.