Structural and expression studies of interferon regulatory factor 8 in Japanese flounder, Paralichthys olivaceus

Interferon regulatory factor 8 (IRF8) plays a role in both innate and adaptive systems in mammals. In this study, the gene and promoter sequences of Japanese flounder, Paralichthys olivaceus, (Po) IRF8 were cloned, and its expression in response to polyinosinic:polycytidylic acid (poly I:C) and lymphocystis disease virus (LCDV) challenges was studied in vivo. The PoIRF8 gene spans over 3.3 kb with a structure of 9 exon-8 intron and encodes 420 amino acids. The putative protein shows the highest sequence identity (69.5-89.0%) to fish IRF8 and possesses a DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS) of vertebrate IRF8. Phylogenetic analysis classified PoIRF8 into the cluster of fish IRF8 within vertebrate IRF8 group of IRF4 subfamily. A number of transcription factor binding sites were identified in the 2348-bp 5' flanking region of PoIRF8 gene, including those of transcription factors for type Ⅰ and type Ⅱ interferon (IFN) inducible genes and genes regulating the development and function of lymphomyeloid cells in mammals. The PoIRF8 transcripts were expressed in all examined tissues of healthy flounders, with higher levels observed in the immune relevant tissues. They were up-regulated by both poly I:C and LCDV treatments in the spleen, head kidney, gills and muscle in an early phase of immune responses, with initiation and peak time points of induction prior to type Ⅰ IFN and Mx. Relative to LCDV, the induction by poly I:C was quicker in all four tissues. These results indicate an involvement of PoIRF8 in the host's antiviral responses and a functional conservation of IRF8 between fish and mammals.