Interaction Of IRF5 And IRF3 Synergistically Up-Regulates IFN Transcription In Ctenopharyngodon Idella

Interferon regulatory factors(IRFs),a family of transcription factors involved in innate immunity,are involved in the transcriptional regulation of interferon(IFN)and interferon-stimulated genes(ISGs).The IRF family consists of nine members of the mammal,10 members of the bird,and 11 members of the teleost fish.Members of the IRF family play important biological roles in antiviral infection,participation in innate and adaptive immune responses,and regulation of apoptosis and differentiation.Among them,the study of interferon regulatory factor 3(IRF3)and interferon regulatory factor 7(IRF7)is more in-depth.In recent years,studies on IRFs have shown that interferon regulatory factor 5(IRF5)can play a role in RIG-I signaling pathway and Toll-like signaling pathway.The research on mammalian IRF5 is mainly focused on its involvement in the regulation of autoimmune diseases.The IRF5 gene in fish is gradually cloned and identified,and the role of fish IRF5 is gradually reported.However,there are few reports on the relationship between IRF5 function in the bony fish and its relationship with the interferon regulatory factor family.Therefore,this study mainly studies the transcriptional regulation of CiIRF5 on IFN,and the regulatory mechanism of CiIRF5 and CiIRF3 synergistically up-regulating IFN,thus further verifying the important role of CiIRF5 and CiIRF3 in fish antiviral immune response.First,we analyzed the stress of grass carp IRF5 gene mRNA expression by detecting lipopolysaccharide(LPS)infection.Real-time quantitative PCR analysis was performed to analyze the expression of CiIRF5 in CO cells after LPS treatment for different time.Among them,CiIRF5 was up-regulated after LPS stimulation for 6h,and reached the highest peak after LPS stimulation for 12 h,then gradually recovered to 0 h level at 48 h;while IFN expression peaked from 6 h,then gradually decreased,and rose at 48 h.It is indicated that Ci IRF5 is involved in the antiviral immune response of cells.In order to study the transcriptional regulation mechanism of grass carp IRF5 in the innate immune signaling pathway,we constructed the grass carp IRF5 eukaryoticexpression vector PcDNA3.1-IRF5,PcDNA3.1-IRF5-nDBD into grass carp cells,and analyzed the expression of IFN by luciferase activity.The results showed that grass carp IRF5 could induce up-regulation of IFN promoter,and IRF5-nDBD was inactive to IFN promoter,revealing the positive regulation of grass carp IRF5 on interferon.To verify that IRF5/IRF3 dimers can synergistically activate IFN expression,we co-transfect grass carp IRF5 and IRF3 into CO cells,and luciferase activity assay to detect the transcriptional activity of IFN promoter.The experimental data showed that the luciferase activity of the IFN promoter of the experimental group co-transfected with CiIRF5 and CiIRF3 was significantly higher than that of Ci IRF5 and CiIRF3,respectively.To further investigate the presence of CiIRF5 and CiIRF3 heterodimers,we performed in vivo immunoprecipitation experiments and in vitro GST-polldown experiments.We constructed CiIIRF5 on a FLAG-tagged plasmid,and CiIIRF3 was constructed on GFP-C1 plasmid and transfected into HEK-293 T cells by transient co-transfection technique.Western blot assay showed that grass carp IRF5 could interact with IRF3.To explore the distribution of grass carp IRF5 in cells,cytoplasmic nuclear protein extraction experiments showed that CiIRF5 was mainly distributed in the cytoplasm,and LPS stimulation could promote the nuclear translocation of Ci IRF5.Cell sublocalization experiments showed that CiIRF5 is localized in the cytoplasm,but the nuclear distribution of CiIRF5 is dynamic.