IDENTIFICATION AND PROMOTER ANALYSIS OF AN IFIT FAMILY GENE FROM ZEBRAFISH DANIO RERIO

IFIT family consists of a group of interferon-induced proteins with Tetratricopeptide Repeat (TPR) motifs. Whereas there are four members in mammals, it is uncertain whether the same IFIT family members exist in fish and whether the fish homologues display the same expression pattern. Our previous report identified two genes from crucian carp Carassius auratus, which are similar to mammalian IFI58 and IFI56 and are induced by grass carp reovirus (GCRV) and interferon (IFN)-containing supernatant. However, unlike mammalian IFI56 gene, virus-induced expression of both genes is blocked by addition of cycloheximide (CHX), a potent inhibitor of protein synthesis. In order to further investigate whether there is an orthologue of mammalian IFI56 in zebrafish, we first searched zebrafish genome data by using mammalian IFI56 sequence. As expected, we identified a hypothetical zebrafish gene, named zebrafish IFIT-A gene, which contained a conserved gene structure similar to mammalian homologues. Zebrafish IFIT-A gene had the largest ORF that encoded a 429-amino-acid protein with conserved TPR motifs. Similar to mammalian IFI56, zebrafish IFIT-A gene was composed of an intron and two exons. RT-PCR showed that zebrafish IFIT-A mRNA was induced in ZFL cell by Poly I:C treatment. Sequence analysis of flanking region of zebrafish IFIT-A gene revealed a typical IFN-stimulated response element (ISRE), which was usually found in the promoters of Interferon Stimulated Genes (ISGs). Consistently, Poly I:C and recombinant crucian carp IFN protein were able to activate the activity of zebrafish IFIT-A promoter. Finally, overexpression of zebrafish IRF3 and IRF7 resulted in a significant increase in promoter activity of zebrafish IFIT-A gene. These data together provided evidence that zebrafish IFIT-A belonged to fish IFIT family and that fish IRF3 and IRF7 might play an important role in regulation of zebrafish IFIT-A expression.