The Mechanism Of PCV2 Ihibits IFN-β Production In PK-15 Cells

Innate immunity is the first line of defense in virus invasion,pattern recognition receptors of host cells detect the invading virus to produce interferons which is one of the most important part of innate immunity.Interferons can limit virus replication and infection,while virus also evolved effective strategies and mechanisms to escape innate immune.Previous studies have showed that the level of the interferons in piglets plasma infected porcine circovirus virus type 2(PCV2)is lower than the swine fever virus or class swine fever virus.Some studies in vitro have also showed that PCV2 can inhibit IFN-α production in dendritic cells and IFN-γ production in peripheral blood lymphocyte.However,until now the mechanisms of PCV2 inhibition of interferon production is still unclear.Hence,PK-15 was used to investigate the mechanism and signal pathway of PCV2 inhibiting interferon production in the study.This will help us to further understand the pathogenic mechanism of PCV2 and the interactions of PCV2 with innate immune.To establish a quicker method to detect the porcine IFN-β expression,the promoter region of porcine IFN-p was amplified from porcine genomic DNA by PCR and cloned into pGL3-basic plasmid.Then the construct was transfected into PK-15 cells and luciferase activities were measured after with or without poly(I:C)stimulation.The luciferase activity obviously increased in PK-15 cells after stimulation with poly(I:C)(1μg//ml)for 12h than control group(P<0.05),indicating that the porcine IFN-β promoter luciferase plasmid works well.This will help us to further investigate the signal pathway of porcine IFN-β.Interferon(IFN)-mediated antiviral response is an important part of host defense.Previous studies reported that PCV2 inhibits interferon production,but the mechanism is still poorly understood.In this study,PK-15 cells were infected with PCV2(MOI=0.1 or MOI=1)for 12h,ISD or poly(I:C)were transfected into PK-15 cells,12h later,IFN-βpromoter activity and mRNA expression level was measured.The results showed that PCV2 significant suppressed IFN-β and IRF3 promoters activities and IFN-β mRNA expression induced by ISD or poly(I:C)(P<0.05),but has no effect on the activation of AP-1 and NF-κB.Furthermore,PK-15cells were infected with PCV2(MOI=1)for 12h,then STING,TBK1,IRF3,or IRF3/5D plasmids were transfected into PK-15 cells,24h later,IFN-β promoter activity was measured.The result showed that PCV2 decreased the IFN-βpromoter activity driven by STING(P<0.05),TBK1(P<0.05),IRF3(P<0.05),and IRF3/5D(P<0.05),these results indicated that PCV2 infection in the downstream of IRF3.PK-15 cells were infected with PCV2,then STING plasmid or ISD were transfected into PK-15 cells,then western blotting and IF,A was applied.The results showed that PCV2 infection caused a reduction in the protein level of nuclear p-IRF3 stimulated by STING,interferes IRF3 nuclear translocation induced by ISD.These results indicated that PCV2 blocks p-IRF3 translocation to the nucleus,thereby inhibiting IFN-β production in PK-15 cells.Our previous studies above have showed that PCV2 inhibits IFN-β production,to examine which ORF encoded by PCV2 participates inhibition of IFN-β production,PCV2 ORF1-4 genes sequences were cloned and inserted into the pcDNA3.1(+)plasmid to obtain ORFs overexpression plasmids.ORFs expression plasmids,IFN-p-Luc plasmid and pRL-TK plasmid were co-transfected into PK-15 cells for 36h,then PK-15 cells were stimulated with ISD,poly(I:C),or IRF3/5D for 12h,dual-luciferase assay was used to measure the IFN-p promoter activity.The results showed that ORF1 significant inhibited ISD,poly(I:C),and IRF3/5D induced IFN-β promoter activity(P<0.05).ORF1-Flag,IFN-β-Luc and pRL-TK plasmids were co-transfected into PK-15 cells with STING,TBK1,IRF3,IRF3/5D,respectively,dual-luciferase assay was used to detect the IFN-β promoter activity after 36h.The results showed that ORF1 significant inhibited STING,TBK1,IRF3 and IRF3/5D induced IFN-β-Luc activity(P<0.05).ORF1-Flag,IRF3-Luc,and pRL-TK plasmids were co-transfected into PK-15 cells for 24h,then stimulated with ISD or poly(I:C)for 12h,IRF3-Luc promoter activity was measured by dual-luciferase assay.The results showed that ORF1 inhibits IFN-β through IRF3 signal pathway.Vector or ORF1-Flag plasmids were transfected into PK-15 cells for 36h,then co-IP was applied to test the interaction of ORF1 with the KPNA3 and KPNA4.The results showed that ORF1 mainly interacted with KPNA3 rather than KPNA4,indicating ORF1 competitively inhibits p-IRF3 interaction with KPN A3.Then,ORF1-Flag ANLS(an ORF1 mutant lacks NLS)or ORF1-Flag plasmids were transfected into PK-15 cells,respectively.co-IP was used to detect the interaction of ORF1-Flag and ORF1-Flag ANLS with KPNA3 after 36h.The results showed that ORF1-Flag interacted with KPNA3,while ORF1-Flag ANLS did not interact with KPNA3,demonstrating that ORF1-Flag ANLS recovered KPNA3 interaction with p-IRF3.Further study showed that ORF1-FlagANLS restored IFN-β-Luc activity inhibited by ORF1,indicating that the NLS is an essential sequence for ORF1 inhibiting IFN-β production.Then,several ORF1 NLS amino acid mutants were constructed and transfected into PK-15 cells respectively,co-IP was applied to measure interaction of these mutants with KPN A3 and KPNA4 after 3 6h.The results showed that the mutant 30K-K-I-R33 was the most crucial amino acid of NLS,the mutant decreased ORF1 combination with KPNA3,and also restored the IFN-β-Luc activity inhibited by ORF1.These results indicate that PCV2 ORF1 NLS combines with KPNA3,then competitively interferes p-IRF3 nuclear translocation,and finally inhibits IFN-β production,which supplies a mechanism of PCV2 evasiving innate immunity and helps us to further understand the interaction of PCV2 and host immunity.