Molecular Cloning Of Porcine E74-like Factor 4(ELF4)and Its Functional Characterization In Activating IFN-?

Innate immunity is the first line for host to defense against the invading pathogens and it is also the foundation of adaptive immunity.The synthesis and secretion of type I interferon(IFN-I)including IFN-? and IFN-? is an important part in natural immunity.In this study,the porcine E74-like factor 4(po ELF4)genes was cloned and its role in IFN-?signaling pathway was investigated.This lays the foundation for the future study of porcine natural immunity.1.Cloning,sequence analysis and tissue expression analysis of porcine ELF4 geneThe multiple sequence alignment of full-length amino acid sequence illustrated that po ELF4 is 91.57% identical to that of the human,and 78.58% identical to that of mouse ELF4 proteins,respectively.Phylogenetic analysis revealed that ELF4 s in mammals are clustered in one group and po ELF4 belongs to a separate evolutionary branch.The expression of po ELF4 transcripts showed that the stomach,small intestine and lymphoid own higher expression,while the highest expression was in small intestine.2.Over-expression of porcine ELF4 significantly activates IFN-?Many molecules in IFN-?signaling pathway are involved in the activation of IFN-?promoter.The po ELF4 eukaryotic expression plasmids were co-transfected into the SIEC?PK-15?IBRS-2 cells with the IFN-?-luc,IRF3-luc luciferase reporter plasmids and internal reference plasmid TK,double luciferase assay confirmed that po ELF4 significantly activated IFN-?-luc and IRF3-luc in a dose-dependent manner.Over-expression of po ELF4 did not promote the nuclear translocation of IRF3.3.The role of different functional domains of po ELF4 for IFN-? activationWe further analyzed the possible functional domain(s)of po ELF4 using the web site and the results showed that po ELF4 contains eight functional domains,thus we generated eight po ELF4 deletion mutants.Western blot showed that all of the eight mutants were able to express normally.Double luciferase experiments the NLS,ETS,53-86 and T2 B deletion mutants reduced the activation of IFN-? promoter,while the absence of other areas had no effect.In previous study,the hu ELF4 360-347 serine/threonine rich domain is indispensable for induction of IFN-? expression,which is responsible for the phosphorylation of hu ELF4.Sequentially,hu ELF4 phosphorylation is essential for the nuclear translocation of hu ELF4,which in turn is required for IFN-? activation.Nuclear localization experiments showed that po ELF4 and most of its mutants were localized in the nucleus,whereas deletion of the 360-347 serine/threonine rich domain in po ELF4 had no effect on its nuclear transportation.Thus,we inferred that there were other phosphorylation sites in this sequence.4.Knockdown of porcine ELF4 blocks Se V-,poly(I:C)-,or poly(d A:d T)-stimulated induction of IFN-?Three pairs of oligonucleotides si RNA specifically targeting po ELF4 were synthesized and the best interference molecules were selected for subsequent experiments.PK-15 cells were transfected with the si NC or po ELF4 si RNA together with an IFN-?-luc reporter plasmid and after 24 h,cells were infected with Se V or transfected with either poly(I:C)or poly(d A:d T).Activation of the IFN-? promoter in cells treated with Se V,poly(I:C),or poly(d A:d T)was attenuated when po ELF4 was silenced by si RNA in a dose-dependent manner.5.A preliminary study on the effect of porcine ELF4 for virus infectionMany molecules in the IFN signaling pathway inhibit the proliferation of virus.In order to analyze the effect of po ELF4 on virus proliferation,PRV and PRRSV were selected as the models of DNA virus and RNA virus,respectively.Different experiments showed that po ELF4 plays an antiviral role during virus infection.In addition,Virus infection(Se V,PRRSV)or immune stimulations(poly(I:C),poly(d A:d T))significantly induce the m RNA expression of po ELF4,whereas PRV infection has no effect on its expression.In conclusion,this study demonstrates that po ELF4 as an important antiviral host restriction factor plays an important role in the IFN-?signaling pathway.