Functional Characterization Of Interferon Regulatory Factor 4(IRF4),5(IRF-5)and Their Roles In The Innate Antiviral Response

IFN regulatory factors(IRFs)are a family of transcription factors that bind a specific DNA motif known as the IFN-stimulated response element(ISRE)and play critical roles in taget genes.The IRFs have crucial biological functions,including the induction of both innate and adaptive immune immunity,antiviral response,proliferation,apoptosis and tumor proliferation.Zebrafish have three members of IRF4,i.e.IRF4 a,IRF4b and IRF4 like gene.Only one IRF5 gene was observed in zebrafish.However,the fuctions of the two IRFs in fish remains poorly understood.Zebrafish was the object in this study,we studied the induction of type I IFN promoter activity by IRF4 a,IRF4b and IRF5 in EPC cell lines.The role of IRF5 was further investigated in respects to the antivial activity and role in downstream signaling pathways.In this study,we constructed eukaryotic expression plasmids for IRF4 a,IRF4b and IRF5 in zebrafish separately.By infection with grass carp reovirus(GCRV)or spring viremia of carp virus(SVCV),IRF4 a,IRF4b and IRF5 transcripts were upregulated in Zebrafish embryonic fibroblast cell line(ZF4).In the ZF4 cell lines,the expression of IRF4 a,IRF4b and IRF5 reached the peak at 12 h after stimulation with GCRV.The expression of IRF4 a,IRF4b and IRF5 were upregulated for 13.8-fold,3.4-fold and 3.3-fold compared with control group respectively.In the ZF4 cell lines,the expression of IRF4 a,IRF4b and IRF5 reached the peak at 24 h after stimulation with GCRV.The expression of IRF4 a,IRF4b and IRF5 were upregulated for 41-fold,3.2-fold and 3.1-fold compared with control group respectively.Then we studied the induction of type I IFN promoter activity by IRF4 a and IRF4 b in zebrafish.Luciferase assay analysis revealed that IRF4 a could induced the activation of type I IFN promoter.Compared with the control group,IFNΦ1 promoter could be activated 5-fold.And IFNΦ3 promoter could be activated 2.3-fold.Luciferase assay analysis revealed that IRF4 b could also induce the activation of IFNΦ1 promoter,but no induction was observed for IFNΦ3 promoter.Compared with the control group,IFNΦ1 promoter could be activated 2.4-fold.Overexpression of DrIRF5 greatly upregulated the expression of Mx,IFN,Viperin and IRF7 in EPC cell lines.Expression of Mx,IFN,Viperin and IRF7 were upregulated by DrIRF5 for 11.7-fold,5.7-fold,54.1-fold and 8.1-fold compared with control group respectively.Then we constructed eukaryotic expression plasmids for FLAG-IRF5,FLAG-IRF5-△DBD and FLAG-IRF5-△IAD,and studied the induction of type I IFN promoter activity by FLAG-IRF5,FLAG-IRF5-△DBD and FLAG-IRF5-△IAD in EPC cell lines.Luciferase assay analysis revealed that IRF5 greatly upregulated the activation of IFNΦ1 promoter.Compared with the control group,IFNΦ1 promoter could be activated 4.4-fold.IFNΦ1 promoter was also significantly induced by IRF5-△IAD,and was activated 3.1-fold compared with the control group.On the other hand,IRF5-△DBD could not activate IFNΦ1 promoter.However,overexpression of IRF5 and IRF5-△IAD inhibited the activation of IFNΦ3 promoter.And IRF5-△DBD had no influence in the activation of IFNΦ3 promoter.IFNΦ3 promoter could be inhibited 2.5-fold and 2.3-fold by IRF5 and IRF5-△IAD respectively compared with the control group.Furthermore,the overexpression of IRF5 in EPC cells could protecte cells from GCRV and significantly inhibited the GCRV virus replication.The determination of Viral Titer revealed that the viral concentration reduced in the overexpression of IRF5 in EPC cells.