Functional Characteristics Of Chicken IRF1 In IFN-? Regulation And Antiviral Innate Immunity

Interferon regulatory factors(IRFs)is an important family for interferon(IFN)expression regulating while viral infection.IRF1,IRF3 and IRF7 are the key regulators for activating type I IFN in mammal,and IRF3 was proved as the master regulator of type-I IFN regulation.While IRF3 is absent in chicken,which makes the regulation mechanism of chicken IFN very unclear.Our previous studies demonstrated that IRF7 could work as IRF3 to compensate the missing of IRF3 to reconstruct Stimulator of Interferon Genes(STING)mediated IFN-? signal pathway.What role does chicken IRF1(chIRF1)play in inducing the production of IFN-?,does chIRF1 and IRF7 promote or restrict each other in the regulation of IFN? These are two scientific problems to be solved in this study.To study the function of IRF1 in IRF3-defected chicken cells,we cloned the chIRF1 gene,synteny analysis indicated that the DNA binding domain was conserved in the course of evolution.Overexpression of chIRF1 activated IFN-? promoter intensively and upregulated the mRNA levels of IFN-? and IFN-stimulated genes(ISGs).Deletion mutant analysis revealed that the transcriptional autoinhibitory domain,DNA binding domain,nuclear localization domain and transactivation domain of chIRF1 are indispensable for IFN-? induction,the third IRF binding site on the IFN-? promoter is the main binding site for chIRF1.Means that IRF1 involved in regulating of IFN-? production.Moreover,the mRNA of chIRF1 was detected with a higher level in immune organs,like spleen,and the mRNA of chIRF1 and IFN-? in different tissues were increase after infection with virus.Implicated that chIRF1 associated with innate immune antiviral response.Overexpression chIRF1 leads the AIV and NDV viral replication suppressed in vitro,to further confirm that chIRF1 is involved in antiviral immune response,by the help of CRISPR/Cas9 system we generated one chIRF1 knockout Chicken DF-1 cell line,the virus replication was increasing in IRF1-lacking cells.Together,these results demonstrate that chIRF1 is an important regulator of IFN-? and is involved in chicken antiviral innate immunity.In wide type DF-1,chIRF1 contributes little in enhancing the ability of STING for IFN-? induction,on the contrary,chIRF1 and STING co-expression in IRF7-lacking cells were activating the IFN-? promoter extremely,and also the chIRF1 in the IRF7-lacking cells.In addition,with the increase of IRF7,the protein amount of IRF1 becomes less.We thus speculate that chIRF1 may be degrade by IRF7 in some way,which interrupt the function of chIRF1 in the STING-depended IFN-? regulatory signaling pathway.Here,we identified chicken IRF1 as a critical IFN-? mediator in response to viral infection,it can interact with IRF7 to participate in the STING mediated IFN-? signal pathway.These investigations offer insights into the mechanism by which chIRF1 responds to viral replication in IRF3-defected chickens.