Molecular cloning and functional characterization of PIASx from Epinephelus coioides reveals its negative regulatory role in antiviral immunity.

The protein inhibitor of activated signal transduction and transcription activation (STAT) (PIAS) family is a key class of signal transduction modulators that regulate innate immune signaling pathways activated by transcription factors including nuclear factor kappa-B (NF-κB), Interferon regulatory factor 3 (IRF3), and Janus kinase/STAT (JAK/STAT). Here, we cloned the PIASx gene from Epinephelus coioides (EcPIASx) and investigated its molecular characteristics. The full-length cDNA of EcPIASx (Accession number: PZ171036) is 1934 bp, containing an open reading frame (ORF) of 1647 bp that encodes a protein of 548 amino acids. Phylogenetic analysis showed that EcPIASx shares the closest evolutionary relationship with Plectropomus leopardus PIASx. EcPIASx is constitutively expressed in all healthy grouper tissues and specifically localized to the nucleus. Overexpression of EcPIASx suppressed type I IFN and NF-κB pathway activation in grouper spleen (GS) cells challenged with SGIV or RGNNV, and downregulated interferon-stimulated genes and pro-inflammatory cytokines. Co-immunoprecipitation and co-localization confirmed EcPIASx interacts with RLR pathway key molecules (MDA5, TBK1, and IRF3). Additionally, EcPIASx upregulated the expression of SGIV and RGNNV genes at both mRNA and protein levels. These results suggest that EcPIASx may negatively regulate antiviral signaling through association with RLR pathway components.