Chicken Anemia Virus VP1 Protein Interferes With IFN-? Expression Via Regulating The CGAS-STING Signaling Pathway

The cyclic GMP-AMP synthase(c GAS)has been identified as an intracellular DNA-sensing of pattern-recognition receptors in the innate immune and mediates downstream immune responses through the stimulator of interferon genes(STING).The c GAS-STING signaling pathway plays an important role in regulating the expression of type I IFN and in combating DNA virus infection.Chicken anemia virus(CAV)infection causes acute and chronic damage and immunosuppression in its host.So far,the molecular mechanisms of how CAV regulates the production of type I interferon through c GAS-STING pathway is still unclear.In the present study,genes of the stimulator molecules in the chicken c GAS-STING pathway were cloned and expressed.The dual-luciferase reporter system of was used to detect the target genes.Moreover,interaction of CAV capsid protein VP1 with stimulator molecules of c GAS-STING pathway signal transduction was investigated on production of IFN-?by cell transfection,immunoprecipitation,and so on.Deeper insight into these interactions might enable us to clarify the mechanisms of CAV infection affecting IFN-?production.1.Gene cloning,protein expression and antibody preparation of c GAS-STING pathway related moleculesCoding region of c GAS,STING,TBK1(TANK-binding kinase 1)and IRF7(Interferon regulatory factor 7)gene were amplified by RT-PCR with specific primers using the c DNA of chicken lymphocyte MDCC-MSB1 as a template,respectively.The fragments amplified were inserted into the procaryotic expression vector p ET32a(+)to construct the recombinant plasmids and then was introduced into the competent cells of E.coli Rosseta.Expression of fusion proteins were induced by IPTG and purified by the gel-cutting method preparing for vaccines.Furthermore,polyclonal antibodies were prepared via immunizing New Zealand white rabbits by subcutaneous injections on multiple sites.The titer of the antibody was determined using the indirect ELISA method.The reactivity between the antibody and the antigen was determined by western blotting.The results showed that the levels of polyclonal antibodies,including anti-c GAS,anti-STING,anti-TBK1 and anti-IRF7 antibodies,were 1:256000,1:512000,1:256000 and 1:512000.These antibodies can effectively recognize the target proteins.2.Lymphocytes infected with CAV induced transcriptional expression of c GAS-STING pathway genesChicken MDCC-MSB1 cells were infected with 10^1.33 TCID₅₀ CAV.The transcriptions of c GAS,STING and IFN-?m RNA were assessed by RT-q PCR in cells in 0 to 24 hours after virus infection.The results showed that the transcriptional levels of c GAS and STING m RNA in the cells were increased significantly at 12 h after virus infection(p<0.001),and then decreased at 24 h.This indicated that c GAS and STING were increased in the early stage of viral infection and was subsequently inhibited by virus.In addition,IFN-?transcription level was suppressed(p>0.05).Our results showed that gene expression of c GAS and STING were significantly activated by CAV infection,while IFN-?expression was inhibited.3.The VP1 of CAV targets IRF7 of c GAS-STING signaling pathway and its key domainTo define which is the target protein of VP1 in the c GAS-STING signaling pathway,the promoter of c GAS,STING,TBK1,IRF7,and IFN-?genes were constructed using chicken DF-1 cell DNA as a template and were cloned into the p GL3-Basic vector to construct the dual luciferase reporter gene detection system.Activity of the gene promoters stimulated by STING overexpression or ISD(interferon stimulatory DNA)were measured in cells The results showed that VP1 protein significantly abolished the activation of IFN-?promoter(p<0.05),significantly inhibit IRF7(p<0.05)but had no any effect on the activity of TBK1 promoter(p>0.05).The results indicated that VP1 could inhibit IFN-?expression signaling pathway by targeting IRF7.To clarify the targeting effect of VP1 on IRF7 and its key domains.p CMV-Myc-IRF7?p CMV-Myc-IRF7-DBD(aa 1-143)and Myc-IRF7-?DBD(aa 143-492)recombinant plasmids were constructed.Futhermore,VP1 and IRF7 or VP1 and IRF7-DBD or IRF7-?DBD were co-overexpressed and co-transfected into HEK293T cells,and the interaction between IRF7 or IRF7 domain and VP1 protein was investigated using Co-IP and Western blotting analysis.The results showed that VP1 co-precipitated with IRF7 and mainly targeted IRF7-?DBD but not IRF7-DBD.This indicates that VP1 regulates the c GAS-STING pathway by binding to IRF7,and IRF7-?DBD is the key domain for its binding.In summary,expression of c GAS,STING,TBK1 and IRF7 proteins of c GAS-STING pathway and their rabbit-derived polyclonal antibodies were prepared.CAV Infection induced c GAS and STING expression in host cells but inhibit the expression of IFN-?.The CAV structural protein VP1 was found to target IRF7 and its?DBD domain and significantly inhibited the expression of IFN-?using a dual luciferase reporter gene system established and immunoprecipitation.Thus,our results clarify the mechanism of VP1interfering the production of IFN-?via interacting with IRF7 of c GAS-STING signaling pathway,which facilitate a better understanding of the mechanisms underlying CAV immune escape.