The Effect Of RLRs And CGAS Signaling Pathways On IFN-? Production And Virus Replication In PCV2-infected PK-15 Cells

Once virus invasion,host cells can recognize the invading virus through the pattern-recognition receptors and induce the innate immune,including the production of antiviral gene,interferon,chemokines and pro-inflammatory cytokines.Surprisingly,in PK-15 cells treatment of IFN-? and IFN-? can enhance the replication of PCV2,which indicating a complex relationship between interferon and PCV2.But there have been no published studies to investigate this potential role of interferon in the context of PCV2 infection.In this study,PK-15 cells were used as a model to explore the effect of PRRs on IFN-? production and virus replication in PCV2-infected PK-15 cells.In order to investigate the effect of RIG-1/MDA-5 signaling pathway on IFN-?production and virus replication in PCV2-infected PK-15 cells,the mRNA level of IFN-?which was detected by real time PCR and IFA in PCV2-infected groups was significant higher than the control at 72h(P<0.01),while treatment of PCV2-infected cells with the IRF3 inhibitor,BX795,the protein of p-IRF3 transfection to nucleus has been inhibited and the expression of IFN-? has decreased(P<0.01),whereas treatment with the NF-TCB inhibitor,BAY11-7082,did not.These findings indicate that PCV2 can induce IFN-?production via the IRF3-mediated rather than the NF-?B-mediated signal pathway.Moreover,PCV2 increased the protein expression levels of MAVS,RIG-1 and MDA-5(P<0.01),and the knockdown of RIG-1 and MDA-5 decreased the expression level of IFN-?in PK-15 cells(P<0.01).Therefore,PCV2 can induce the IFN-? production via the RIG-1/MDA-5-MAVS-IRP signaling pathway.Furthermore,the PCV2 load and PCV2 infectivity significant decreased after knockdown of RIG-1 and MDA-5,which was detected by real time PCR and IFA,respectively,indicating that PCV2 can induce the interferon through RIG-1 and MDA-5 signaling pathways,which can contribute to PCV2 replication.In conclusion,PCV2 induces the production of IFN-? via the RIG-1 and MDA-5 signaling pathways,and the IFN-? produced during PCV2 infection facilitates viral replication.Currently,different classes of PRR families have been identifiied and cGAS is the most important DNA senser and it express in diverise cellular.Senser cGAS mainly recognizes viral DNA and induces the production of interferon through the STING/IRF3 signaling pathway.In order to investigate the effect of cGAS-STING signaling pathway on IFN-?production and virus replication in PCV2-infected PK-15 cells,real time PCR were used to detect the mRNA level of cGAS and STING in PK-15 cells following PCV2 infection for 24,48 and 72 h.The results showed that the mRNA expression level of cGAS in the PCV2-infected group were significantly higher than control at 24,48 and 72 h(P<0.01),and the level of STING were only significantly higher than control at 72 h(P<0.01).The western boltting result also showed that the protein expression level of STING were significant higher than control at 72h after PCV2 infected.The results Native PAGE and immunofluorescence indicating that the dimerization and nuclear translocation of STING in PCV2-infected groups are significant higher than the control at 72h(P<0.01).In addition,knockdown of cGAS or STING decreased the expression level of IFN-? in PK-15 cells after PCV2-infected(P<0.01).Furthermore,the PCV2 load and PCV2 infectivity decreased after knockdown of cGAS or STING,which was detected by real time PCR and IFA,respectively,indicating that cGAS and STING signaling pathways contribute to PCV2 replication.In conclusion,PCV2 can induce the production of IFN-? via the cGAS and STING signaling pathways,and the IFN-p produced during PCV2 infection facilitates viral replication.In conclusion,PCV2 induces the production of IFN-? via the RIG-1/MDA-5-MAVS and cGAS-STING signaling pathways.The IFN-? expression level and the PCV2 load and infectivity were decreased after knockdown of RIG-1/MDA-5 or cGAS/STING.