| Host cells recognize the pathogenic microorganism through the pattern recognition receptors,and then induce the production of interferon through the cascade amplification mechanism.cGAS is the most important cytoplasmic DNA recognition receptor.It can synthesize cGAMP after recognizing pathogenic DNA.STING is activated by cGAMP and then recruits TBK1 and IRF3 to form a ternary complex,finally induces IRF3 phosphorylation,dimerization and nucleation to produce type I interferon.However,many DNA viruses have evolved a variety of strategies to escape the antiviral innate immune response,which is related to the signaling pathway.Our previous experiments have shown that PCV2 can inhibit the production of interferon induced by ISD and Poly I:C,but it is not clear which proteins encoded by PCV2 can play an inhibitory role in the cGAS/STING signaling pathway,and the specific mechanism also has not been reported.Based on this,we screened the encoded proteins and investigated their roles in the cGAS/STING signaling pathway to understand the molecular mechanism of PCV2 pathogenicity.In the experiment,we first constructed 4 PCV2-encoding protein expression plasmids.The promoter activity and the mRNA level of IFN-β showed that the PCV2 Rep significantly inhibited the production of IFN-β mediated by cGAS-STING(P<0.05).The promoter activity of IRF3 and the promoter activity and the mRNA level of IFN-β showed that PCV2 Rep targeted IRF3 or its downstream to inhibit the production of IFN-βmediated by cGAS-STING(P<0.05).IFA showed that PCV2 Rep inhibited IRF3 activation,and further experiments showed that Rep inhibited IRF3 phosphorylation and IRF3/5D entry into the nucleus,but did not inhibit IRF3/5D dimerization.These results indicated that PCV2 Rep blocked the processes of IRF3 phosphorylation and entry nuclear.Co-IP showed that Rep only interacted with KPNA3 and blocked the binding of KPNA3 and IRF3/5D.In addition,the promoter activity of IFN-β also proved that overexpression of KPNA3 can significantly restored Rep-inhibited IRF3/5D-induced IFN-β promoter level(P<0.05).These results indicated that PCV2 Rep blocked IRF3/5D from entering the nucleus by targeting KPNA3,thereby inhibiting the production of IFN-β.The Rep△NLS plasmid was constructed on the basis of the Rep expression plasmid.Co-IP showed that Rep△NLS weakened the binding ability of KPNA3 and IRF3/5D.In addition,the promoter activity and the mRNA level of IFN-β also showed that Rep△NLS inhibited IFN-β induced by cGAS-STING production was weakened(P<0.05).These results indicated that NLS is a key sequence for PCV2 Rep to inhibit the binding of KPNA3 to IRF3/5D and the production of IFN-β.The 6 expression plasmids of Rep NLS mutants were constructed by successively mutating 4 or 6 amino acids into inactive alanine.The promoter activity of IFN-β showed that 22NNPS25 and 30KKIR33 mutants have the weakest ability to inhibit the production of IFN-β induced by cGAS-STING and IRF3/5D(P<0.05),indicating that they were key amino acid sequences to inhibit the production of IFN-β.These results indicated that the PCV2 Rep protein inhibited the binding of KPNA3 and IRF3/5D through interaction with KPNA3,thereby inhibiting the phosphorylation of IRF3 into the nucleus and the production of IFN-β induced by cGAS-STING,22NNPS25 and 30KKIR33 in Rep NLS were key amino acid sequences.Previous experiments have shown that PCV2 Rep inhibited the production of IFN-β by targeting KPNA3,but whether KPNA3 affects PCV2 replication is unclear.In this experiment,the effects and molecular mechanisms of KPN A3 on PCV2 replication in PK-15 cells were explored through RNA interference and overexpression experiments.Knockdown of KPNA3 and infection with PCV2,cells were collected after a certain period of time for detection.The PCV2 infectivity,the PCV2 load and the expression level of PCV2 Cap increased significantly at 48 and 72 h,which were detected by IFA,qRT-PCR and Western Blotting,respectively(P<0.05);and the PCV2 infectivity,the PCV2 load and the expression level of PCV2 Cap decreased significantly after overexpressing KPNA3(P<0.05).The mRNA levels of IFN-β,ISG54,Mxl and ISG56 decreased significantly at 48 and 72 h when knockdown of KPNA3(P<0.05),while the mRNA levels of these genes increased significantly when overexpressing KPNA3(P<0.05);when extracting cytoplasmic nuclear proteins,it was also found that knockdown of KPNA3 inhibited IRF3 from entering the nucleus,and overexpression of KPNA3 promoted IRF3 to enter the nucleus.At the same time,knockdown of KPNA3 promoted p65 to enter the nucleus,while overexpressing KPN A3 was the opposite;and the mRNA levels of IL-10 and IL-1βsignificantly increased at 72 h when knockdown of KPNA3(P<0.05),while the mRNA levels of these genes significantly decreased when overexpressing KPNA3(P<0.05).These results indicated that knockdown of KPN A3 promoted the replication of PCV2 in PK-15 cells,while overexpressing KPNA3 inhibited the replication of PCV2.This was because KPNA3 promoted IRF3 to enter the nucleus and induced an antiviral response,thereby inhibiting PCV2 replication and NF-κB signal pathway.In conclusion,the PCV2 Rep protein interacted with KPNA3 to block the binding of KPNA3 and IRF3/5D,thereby inhibiting the phosphorylation of IRF3 into the nucleus and the production of IFN-β mediated by cGAS-STING,and 22NNPS25 and 30KKIR33 in Rep NLS were key amino acid sequences.Knockdown of KPNA3 promoted the replication of PCV2 in PK-15 cells,and overexpression of KPNA3 inhibited the replication of PCV2,this was because KPNA3 promoted IRF3 into the nucleus and induced an antiviral response,thereby inhibiting PCV2 replication and NF-κB signal pathway. |
