Antiviral Protection Mechanisms Mediated by IFNγ Isoforms Through 2 Distinct IFNγ Receptor‐STAT1 Signaling Pathways in Fish

The antiviral activity and signal transduction mechanism corresponding to the IFNγ‐JAK‐STAT signaling pathway found in humans and mice have never been characterized, although fish genomes possess 3 distinct genes of type II IFN, i.e. IFNγ1, IFNγ2, and IFNγ‐related (IFNγrel). To reveal the antiviral functions and signaling pathways of the type II IFN system in the ginbuna crucian carp, Carassius auratus langsdorfii , we produced recombinant IFNγ1, IFNγ2, and IFNγrel proteins in bacteria, and found them to exhibit high antiviral activities against crucian carp hematopoietic necrosis virus; their ED 50 were at 25 ng/mL. We also cloned 2 distinct IFNγ receptor alpha chain (IFNGR1) isoforms, IFNGR1‐1 and IFNGR1‐2, and stably expressed them in HeLa cells by transfecting the cells with IFNGR1‐1 or IFNGR1‐2 cDNA. When the receptor cDNA‐transfected cells were treated with the ligands in a 1 ligand‐on‐1 receptor manner (IFNγ1 and IFNGR1‐2 or IFNγ2 and IFNGR1‐1), the STAT1 protein was phosphorylated at both serine‐727 and tyrosine‐701 residues. Gel shift mobility analysis together with a reporter assay clearly showed that the specific ligand‐receptor interaction resulted in the binding of the STAT1 protein to the GAS (IFNγ activated site) element and in transcriptional enhancement in ginbuna crucian carp GTS9 cells. Therefore, both IFNγ1 and IFNγ2 actions were found to be mediated by a specific receptor for each signaling via a STAT1‐dependent mechanism in the fish.