Expression Of IFN-β Regulated By Chicken Anemia Virus VP2 Protein Via Targeting CGAS-STING Signaling Pathway

CyclicGMP-AMP synthase(cGAS)plays an important role in recognizing cytoplasmic DNA to activate type I interferon production to regulate antiviral innate immune responses.Our previous study found that the capsid protein VP1 of chicken anemia virus(CAV)inhibited the expression of IFN-β by targeting IRF7 of the cGASSTING(stimulator of interferon genes)signaling pathway.It is unclear whether VP2 which assissts the expression of VP1 plays a similar role.This study intends to explore the molecular pathway of CAV-VP2 regulating IFN-β expression by targeting cGASSTING signaling pathway,and will provide the basis for further revealing the molecular mechanism of CAV regulating IFN-β expression.1.VP2 enhances transcriptional activity of IFN-β gene.In this study,the eukaryotic expression plasmid pm Cherry-C1-VP2 was constructed and transfected into chicken macrophages HD11.The transcription levels of VP2 and IFN-β genes at 0 to 36 h after transfection were detected by relative real-time quantitative PCR.The eukaryotic expression plasmid p CMV-Myc-VP2 and IFN-βpromoter expression plasmid(IFN-β-luc)were co-transfected into HD11 cells,and the co-transfection of p CMV-Myc-VP1 plasmid and IFN-β-luc was used as a reference.Under the stimulation of interferon stimulatory DNA(ISD),the promoter activity of IFN-β gene under VP2 expression was analyzed by double luciferase reporter system.The results showed that after over-expression of VP2,the IFN-β m RNA level in the cells was significantly up-regulated and reached the extremely significant level at 24 to36 h.It indicated that VP2 could enhance the transcriptional expression of IFN-β.Contrary to the effect of VP1,VP2 significantly promoted the initiation activity of IFN-β gene.It is necessary to further analyze what signaling molecules VP2 acts on cGASSTING pathway to regulate IFN-β production.2.VP2 activates related molecules in cGAS-STING signaling pathway.In order to further explore the related molecules in cGAS-STING signaling pathway interacting with VP2,p CMV-Myc-VP2 plasmid was transfected into chicken fibroblasts DF-1.The effects of VP2 on the activities of cGAS promoter(cGAS-luc),STING promoter(STING-luc),TBK1 promoter(TBK1-luc)and IRF7 promoter(IRF7-luc)in cGAS-STING signaling pathway were detected by the dual luciferase reporter system.The results showed that VP2 significantly activated cGAS-luc and STING-luc,but not TBK1-luc and IRF7-luc.This suggests that the target molecules regulated by VP2 may be cGAS or STING.3.VP2 promotes IFN-β expression by direct regulating cGAS.The eukaryotic expression recombinant plasmid p CMV-HA-VP2 was constructed and co-transfected with p CMV-Myc-cGAS or p CMV-Myc-STING into HEK293 T cells.Co-IP analysis showed that VP2 could be combined with cGAS or STING and coprecipitation occurred.Then pm Cherry-C1-VP2 was co-transfected with p EGFP-C1-cGAS or p EGFP-C1-STING plasmid,indicating that VP2 only colocalizated with cGAS.VP2 was used as a bait protein,and Pull-down analysis showed that VP2 binds cGAS but not STING.Therefore,cGAS is the target molecule of VP2 in cGAS-STING pathway.VP2 and cGAS were overexpressed,and IFN-β protein level was detected by double-antibody sandwich ELISA.The results showed that co-transfection with VP2 and cGAS could significantly up-regulate IFN-β,compared with cGAS transfection alone.This indicated that VP2 could promote the expression of IFN-β induced by cGAS.In summary,our results confirmed that VP2 targeted cGAS to regulate cGAS-STING signaling pathway to promote IFN-β expression.The results will lay a foundation for further revealing the mechanism of the effect of cGAS-STING signaling pathway regulated by different encoded proteins of CAV on IFN-β expression.