Mechanisms Of Inhibition Of IFN-? Induction And Its Down Stream Signaling By Porcine Circovirus Type 3 Cap

Type I interferons(IFN-I)are one type of the most important cytokines in mammalian innate antiviral immunity.IFN-I defenses against viral infection through activating JAK-STAT signaling to induce interferon-stimulated genes(ISGs)expression.In order to facilitate the replication of themselves,viruses evolve diverse strategies to evade the host IFN-I mediated innate immunity.Therefore,it will help for us to understand the pathogenic mechanism of the virus to study the mechanisms of how viruses evade host innate immunity,which is also better for viral diseases prevention and control and vaccines development.Porcine Circovirus Type 3(PCV3)is a novel PCV discovered in recent years,which might associate with the clinical diseases of sow reproductive failure,respiratory disease,porcine dermatitis and nephrotic syndrome(PDNS),carditis etc.Since the first report of PCV3 in the pig herd with PDNS and sow reproductive failure in USA in 2016,PCV3 was subsequently detected in suspected-PCVAD pig herd in countries in Asia,South American and Europe,which causes a wide attention in pig industry.However,the pathogenicity and pathogenic mechanisms of PCV3 remain unclear until now.In the recent study,we investigated the prevalence of PCV3 in south China and discovered the mechanisms of the inhibition of IFN-I mediated innate immunity by PCV3 Cap,which would provide theoretical basis for the study of PCV3 pathogenicity.To study the prevalence situation of PCV3 in 2018 in south China,total 163 samples from 55 pig farms suspected with PCVAD were collected for PCV3 and PCV2 detection by PCR.The results showed that the positive rate was 35.58% for PCV3 genome and 47.24% for PCV2 genome,and the co-ipositive rate of PCV3 and PCV2 genome was 23.31%,which demonstrated high prevalence of PCV3 and PCV2 in south China.Phylogenetic analysis basic on PCV3 complete genome showed that PCV3 could be divided into PCV3 a and PCV3 b group,and both tow group strains circulated in south China.The codon optimized PCV3 ORF2 were inserted into p Cold-TF and p ET-28 a vector and transformed into E.coli BL21(DE3).SDS-PAGE analysis showed that a 76 k Da TF-Cap and a 26 k Da His-Cap protein were successful expressed after IPTG induction.The TF-Cap was used as antigen to immunize BALB/c mice 5 times.The spleen cells from immunized mice were fused with Sp2/0 myeloma cell.Eight monoclonal cell strains were sorted out by subclone and ELISA assay.Two monoclonal antibodies were prepared and purified using tow monoclonal strains,3-2-3-6-2 and 3-21-4-6-2.The titer of both two antibodies are over 1:8000,which might provide a material basis for PCV3 research.In order to investigate the effect of PCV3 Cap on host innate immunity,we firstly study the mechanism of that Cap inhibits IFN-? induction by DNA.We found that Cap significantly suppressed ISD induced IFN-? m RNA transcription when conducting real-time PCR.When conducted dual-luciferase assay,we found that Cap significantly inhibited IFN promoter activation induced by ISD and Poly(d A:d T)and Cap inhibited IFN promoter activation induced by ISD at dose-dependent manner.Western blotting analysis showed that Cap inhibited TBK1 and IFR3 activation by ISD.And further investigation showed that Cap did not affect IFN promoter activation induced by overexpression of c GAS and STING.We also found that Cap inhibit c GAS binding to ISD,which show that Cap inhibits DNA induced IFN production at the DNA binding of c GAS.We found that G3BP1 interacts with Cap when conducting pull down and LC/MC and Cap inhibited the interaction between G3BP1 and c GAS.The activation of IFN promoter and phosphorylation of TBK1 induced by ISD were significantly reduced when the G3BP1 was knocked down by si RNA,which demonstrated that G3BP1 involes in IFN indction by DNA.Overexpression G3BP1 attenuate the inhibition ISD binding of c GAS by Cap and promoted phosphorylation of TBK1 and IRF3 induced by ISD.Taken together,Cap inhibits DNA induced IFN-? production through interaction with G3BP1.Further study that the effect of Cap on the IFN-? induced ISG was carried out and the results showed that Cap significantly inhibited IFIT1 and IFIT2 transcription and ISRE promoter activation induced by IFN-?,but Cap did not affect the protein level and phosphorylation of STAT1 and STAT2 and as well as the dimerization and nuclear translocation of STAT1 and STAT2.Subsequently,we found that Cap significantly inhibited IRF9-S2 C,an ISGF3 mimic,activating ISRE promoter,which demonstrated that Cap might inhibit ISRE promoter bind of IRF9 or transcriptional activation of STAT2.We further found that Cap interacts with STAT2 but not STAT1 or IRF9,and the interaction domain of STAT2 interacting with Cap is the C-terminal transcription activation domain(TAD),which demonstrated that Cap inhibits ISGF3 transcriptional activity through interaction with the TAD of STAT2.In addition,we found that Cap could binding to ISRE and inhibit ISRE binding of IRF9-S2 C when conducted biotin-ISRE pull down.In conclusion,the investigation of PCV3 prevalence in south China in this study provides an information support for PCV3 prevention and control.We also illuminate the molecular mechanism that Cap inhibits IFN-?-mediated innate immunity,which provides scientific basis for the pathogenesis research of PCV3.