Molecular Mechanism Of Peste Des Petits Ruminants Virus ? Protein Inhibits RLRs-mediated Type ? IFN Signaling Pathway

Viruses use different strategies to avoid immunosurveillance that include the induction of immunosuppression,maximize its chances of survival,replication and transmission,the clinical consequences on the host may be severe,and induce secondary infection.Therefore,understanding the mechanisms involved in the induction in immunosuppression is crucial for controlling important human and animal viral infection.Pete des petits ruminants(PPR)is an important infectious disease caused by Pete des petits ruminants virus(PPRV),which mainly infects small ruminants such as sheep and goats.PPR is listed as a disease that must be reported by the Office international des epizooties because of its extremely high morbidity and mortality in sheep-raising industry.The aim of the present study is to investigate the mechanism of PPRV non-structural V protein in innate immunity,The Real-time fluorescence quantitative PCR(qPCR)is used to detect the effect of V gene on the expression of Interferon(IFN)and its downstream factors mediated by the Retinoic-acid-inducible gene(RIG-I)like receptor(RLRs)signaling pathway at the transcription level;the dual-luciferase reporter gene assay is used to detect the target of V protein in RLRs pathway;qPCR and Western blot(WB)are used to further study the antiviral effect of V protein on endogenous and exogenous IFN mediated.WB is used to study the effect of V protein on the phosphorylation of Interferon regulatory factor 3(IRF3)and Signal transducers and activators of transcription(STAT1).Finally,the full-length cDNA cloning of PPRV and full-length cDNA cloning of V gene mutation are constructed.The results are as follows:1.PPRV V protein inhibits RLRs pathway-mediated the generation of IFN-? and the expression of its downstream cytokines.Co-transfection of pRK-Flag-RIG-IN and pcDNA3.1-HA-V to HEK293 T cells,qPCR detection shows that PPRV V protein most significantly down-regulated RLRs pathway-mediated the production of IFN-?,and its Interferon stimulated gene 56(ISG56),ISG15,C-X-C motif chemokine 10 gene(CXCL10)in the expression of gene transcription level(p<0.001).2.The detection of dual-luciferase reporter gene assay shows that the overexpression of PPRV V protein in the cells can most significantly inhibit RLRs pathways the activity of Interferon-stimulated response elements(ISRE)(p<0.001)reporter gene in the RLRs pathway,indicating V protein can inhibit the production of IFN in RLRs pathways.3.PPRV V protein inhibits the antiviral effects of HEV,PPRV and EMCV via decreasing production of their endogenous IFN response and exogenous IFN response.pcDNA3.1-HA-V and pRK-Flag-RIG-IN are co-transfected into HEK293 T cells and Huh7-p6 cells for 24 h,the supernatant is collected and added to the newly cultured cells for furthercultured 24 h,the HEK293 T cells are sampled in the 12 h,24 h and 36 h inoculated by PPRV and Encephalomyocarditis virus(EMCV),in the Huh7-p6 cells 18 h and 36 h inoculated by Hepatitis E virus(HEV).The results of qPCR shows that PPRV V gene can most significantly increase the replication of HEV at 18 h(p<0.001)and no influence at 36 h(p>0.05),most significantly increase the replication of PPRV and EMCV at the three time period(p<0.01),indicating PPRV V protein can inhibit the antiviral effects of endogenous IFN.Co-transfection pcDNA3.1-HA-V and pRK-Flag-RIG-IN to HEK293 T cells and Huh7-p6 cells for 24 h,commercial IFN-?(1000 U/mL)and IFN-?-2b(1000 U/ml)are added for culturing for 24 h,the results of qPCR and WB shows that V protein significant increase the m RNA expression of three viruses(p<0.05),indicating that PPRV V protein can significant inhibit the antiviral effect of exogenous IFN.4.PPRV V protein inhibits antiviral effect of IFN-I through blocking JAK-STAT pathway.WB shows that transfection of pRK-Flag-RIG-IN in HEK293 T cells enhances the phosphorylation of IRF3,while pcDNA3.1-HA-V decreases the phosphorylation of p369-IRF3 and p701-STAT1,indicating V protein inhibits the phosphorylation of IRF3 and STAT1,reduces the production of IFNs,blocks the signal transmission in JAK-STAT signaling pathway and destroies the host antivirus states.5.The 16153 bp of PPRV full-length cDNA clone and V gene mutation full-length cDNA clone are constructed.According to the conservation of the PPRV genome sequence,the Hammerhead ribozyme sequence(HamRz)introduce at the 3' end of the genome and the T7 terminator and the Hepatitis delta virus ribozyme sequence(HdvRz)introduce at the 5' end,respectively,7 fragments are cloning separately by RT-PCR and connect to each other to obtain the PPRV full-length cDNA clone(pBluescript SK-PPRV)and V gene mutation full-length cDNA clone(pBluescript SK-PPRV?V).The present study analyzes the natural immune mechanism of PPRV non-structural protein V mediated in host cells,clarifies the basic molecular mechanism of V protein can inhibit RIG-I-mediated IFNs production in RLRs pathway,which makes conclusion that V protein inhibits the phosphorylation of STAT1 and IRF3 in the JAK-STAT pathway to produce the antiviral effect of IFN,full-length cDNA clones of PPRV and full-length cDNA clones of V gene mutations are constructed,these findings lay the foundation for further elucidating the natural immune mechanism mediated by non-structural proteins of PPRV.