Molecular Mechanism Of Peste Des Petits Ruminants Virus(PPRV) Inhibits Type ? IFN Signaling Pathway

Peste Des Petits Rumminants(PPR)is an acute infectious disease caused by Peste Des Petits Rumminants Virus(PPRV),which mainly infects small ruminants.PPR has brought serious harm to sheep industry because of its high morbidity and mortality.In the present study,to investigate the PPRV C-mediated innate immune responses,real-time fluorescent quantitative PCR(qPCR)was used to detect the effect of C gene on interferons(IFNs)and their downstream factors in retinoic-acid-inducible gene I-like receptor(RLRs)pathway at transcriptional level.A dual-specific luciferase reporter assay was performed to test the interaction between C with the key molecules in the RIG-? signaling pathway.More experiments were done to analyze the effect of C protein on antiviral state mediated by endogenous and exogenous IFN,the signal transducers and activators of transcription I(STAT1)also be tested.? The following results were obtained:1.Co-transfection of pRK-Flag-RIG-IN and pCMV-HA-C to HEK293 T cells,the results from qPCR indicated that PPRV C protein inhibited the production of INF-? mediated by RLRs signaling pathway and the expression of interferon stimulated gene 15(ISG15),ISG56 and CXC chemokine ligand 10(CXCL10)at gene transcription level.2.On the basic,HEK293 T cells were transfected with internal reference plasmid pRL-TK and reporter gene plasmid pGL3-IFN-?,pGL3-ISRE,pGL3-NF-?B,respectively.Than the experimental group co-transfected pRK-Flag-RIG-IN and pCMV-HA-C,the negative control group co-transfected pCMV-HA,the positive control group co-transfected pRK-Flag-RIG-IN.The results of A dual-specific luciferase reporter assay showed that the overexpression of PPRV C protein could significantly inhibit the activity of IFN-?(p < 0.001),NF-?B(p < 0.01)and ISRE(p < 0.001)reporter genes mediated by RIG-IN in RLRs pathway.The results of more experiment showed that the overexpression of PPRV C protein significantly inhibited the activity of IFN-? promoter activated by RIG-IN-mediated mitochondrial antiviral signaling(MAVS)in RLRs pathway in a dose-dependent manner(p < 0.05).The activities of I-kappa kinase(IKK?),TRAF family member-associated NF-?B activator(TANK)-binding kinase 1,TBK1,tumor necrosis factor receptor associated factor 3(TRAF3),interferon regulatory factor 3(IRF3),IRF7 were not significantly inhibited by PPRV C(p ? 0.05),All data suggest that the target of C protein in RLRs pathway3.After co-transfection of pCMV-HA-C and pRK-Flag-RIG-IN to HEK293 T cells for 24 hours,supernatants were collected and added to Freshfully HEK293 T for 24 hours.Than HEK293 T were infected with PPRV,Encephalomyotic Virus(EMCV)and Vesicular Stomatitis Virus(VSV),respectively.The results of qPCR showed that PPRV C protein could significantly increase mRNA expression of the three viruses in HEK293T(p < 0.001).After co-transfection of pCMV-HA-C and pRK-Flag-RIG-IN to HEK293 T for 24 hours,treated by commercial IFN-?(1000 U/mL)24 hours and then infected with three viruses respectively.Quantitative PCR showed that C protein also significantly increase the mRNA expression of the viruses(p < 0.001).All date indicate that PPRV C protein could significantly inhibit the endogenous and exogenous IFN to break the antiviral state.4.At experimental group and control group,pCMV-HA and pCMV-HA-C was transfected into HEK293 T respectively,than IFN-?(1000U/mL)was used to activate the cellular IFN response pathway.The results of western blot analysis showed that there was no difference in the expression of STAT1 between experimental group and control group,but the expression of P-STAT1 in experimental group decreased.These data suggest that PPRV C protein can inhibit STAT1 phosphorylation,but the mechanism of PPRV C protein blocking JAK-STAT signal transduction to inhibit the antiviral state of interferon still needs further study.The studied of innate immune mechanism mediated by PPRV non-structural protein C in host cells,indicated the basic molecular mechanism that C protein can inhibit RIG-I-mediated IFNs production and signal transduction in RLRs pathway,demonstrated that C protein can inhibit the broad-spectrum antiviral effect of IFNs,and preliminarily concluded that the inhibition of STAT1 phosphorylation in JAK-STAT pathway by C protein is the reason for its antiviral effect on IFN production.These data lay a foundation for further elucidating the innate immune mechanism mediated by PPRV non-structural protein.