Interaction Of IRF9 And STAT2 Synergistically Up-regulates IFN And PKR Transcription In Ctenopharyngodon Idella

IRF9 is a key factor in the type I IFN signalling.Under the stimulation of type I IFN,IRF9 interacts with STAT1 and STAT2 to form the IFN-I-stimulated gene factor3(ISGF3)which activates the transcription of ISGs.However,many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells.In the present study,the fulllength cDNA sequence of IRF9(termed CiIRF9,KT601055)and STAT2(term CiSTAT2,KT781914)from grass carp were cloned and identified.The fulllength of cDNA sequence of CiIRF9 contained a1287 bp of ORF,a 731 bp of 3'UTR and a 49 bp of 5'UTR sequence.The fulllength of cDNA sequence of CiSTAT2 contained a 2550 bp of ORF,a 241 bp of 3'UTR and a122 bp of 5'UTR sequence.A low level of constitutive expressions of CiIRF9 and CiSTAT2 were detected by RT-PCR in grass carp tissues(brain,eye,gill,skin,liver,spleen,kidney and intestine).After poly I: C stimulation,CiIRF9 mRNA was obviously up-regulated in all tested tissues and the highest was in liver tissue and CiSTAT2 mRNA was obviously up-regulated in all tested tissues and the highest was in intestine tissue after 48 h stimulation.After LPS stimulation,CiIRF9 mRNA was obviously up-regulated in all tested tissues except in skin and the highest was in liver tissue after 24 h stimulation.After LPS stimulation,CiSTAT2 m RNA was obviously up-regulated in all tested tissues except in eye and the highest was in liver tissue after24 h stimulation.In vitro,a high-affinity interaction between Ci IRF9 and the promoter of CiIFN or CiPKR was demonstrated by gel mobility shift assay.Results shown that CiIRF9 displayed a distinct resistance to the move of IFN and PKR promoters,On the contrary,CiIRF9-nDBD had no trailing phenomenon.In vivo,the promoter activities of IFN and PKR were increased by transient transfection of CiIRF9 but were not by transient transfection of CiIRF9-nDBD.The results indicated that CiIRF9 was involved in up-regulation of IFN and PKR gene transcription and the N terminal of CiIRF9 was inportant in the regulation.In order to the dimmer IRF9/STAT2 acts as the ISGF3 activated the transcription of ISGs in grass crap,the co-transfection of CiIRF9 and CiSTAT2 were performed.co-transfection of CiIRF9 and CiSTAT2 was able to regulate IFN and PKR promoter activity more significantly compared to transfection of CiIRF9 or CiSTAT2 alone.Moreover,the interaction of CiIRF9 and CiSTAT2 was further investigated by in vivo and in vitro protein interaction assays.Recombinant CiIRF9 and CiSTAT2,both tagged with FLAG(or HA),were expressed in HEK 293 T cells by transient transfection experiment.Co-immunoprecipitation assays showed that CiIRF9 can interact with CiSTAT2 in vivo.Soluble ST2-936-GST(containing the N-terminal and coiled-coil domain of CiSTAT2)was expressed and purified from E.coli.A GST pull-down assay suggested that GST-tagged ST2-936 efficiently bound to FLAG-tagged IRF9.