IRF-1 acts as a positive regulator in the transcription of grass carp (Ctenopharyngodon idella) IFN gene

Abstract

Interferon regulatory factors (IRFs) are well-known to be crucial for modulating the innate immune responses to viral infections. In the present study, the IRF-1 gene of grass carp (Ctenopharyngodon idella) (termed CiIRF-1) was cloned and characterized. The complete genomic sequence of CiIRF-1 was 3150 bp in length and comprised 9 exons and 8 introns. The CiIRF-1 promoter sequence was 558 bp in length. The largest open reading frame (ORF) of the full CiIRF-1 cDNA sequence was 870 bp, and encoded a polypeptide of 289 amino acids. The putative CiIRF-1 was characterized by a conserved N-terminal DBD (113 aa), and included a signature of five conserved tryptophan residues. Phylogenetic relationship analysis revealed that CiIRF-1 was highly homologous to the counterparts of other teleosts and mammalians. CiIRF-1 was expressed at a low constitutive level but was significantly up-regulated following stimulation with either Poly I:C or recombinant grass carp (C. idella) IFN (rCiIFN) in all 6 tested tissues, especially in spleen and gill. The recombinant CiIRF-1 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. Three different fragments of promoter sequences from grass carp type I IFN (CiIFN) gene (GU139255) were amplified. These fragments included the proximal region (CiIFNP2), the distal region (CiIFNP6), and the full length of CiIFN promoter sequences (CiIFNP7). Gel mobility shift assays were employed to analyze the interaction between CiIRF-1 and CiIFN promoter sequences. The results revealed that CiIRF-1 could bind to CiIFN promoter with high affinity in vitro. Subsequently, the recombinant plasmid of pGL3-CiIFNPs and pcDNA3.1-CiIRF-1 were constructed and transiently co-transfected into C. idella kidney (CIK) cells. The impact of CiIRF-1 on CiIFN promoter sequences were measured by luciferase assays. These results demonstrated that CiIRF-1 acts as a positive regulator in the transcription of grass carp IFN gene.