Functional characterization of IRF2 and IRF2a in Japanese eel (Anguilla japonica) identifies dual pathways...

Interferon regulatory factor 2 (IRF2) is a transcription factor that critically modulates innate antiviral immunity through regulation of interferon (IFN). In this study, two IRF2 homologs, AjIRF2 and AjIRF2a, were identified and characterized from Japanese eel (Anguilla japonica). Sequence analysis revealed that both proteins harbor a highly conserved DNA-binding domain (DBD). Expression analysis demonstrated that AjIRF2 and AjIRF2a are ubiquitously expressed across tissues, with AjIRF2a exhibiting consistently higher basal expression, particularly in the kidney. Upon infection with Anguillid herpesvirus 1 (AngHV-1), as well as stimulation with poly I:C, both AjIRF2 and AjIRF2a were markedly upregulated. Functional analyses showed that AjIRF2/2a attenuate the induction of AjIFNs and interferon-stimulated genes triggered by AngHV-1 or poly I:C, thereby facilitating viral replication. Dual-luciferase reporter assays further demonstrated that AjIRF2/2a antagonize AjIRF1- and AjIRF11-mediated activation of the AjIFN promoter by competing for interferon-stimulated response element (ISRE) binding, which depends on four key residues (Arg82, Cys83, Asn86, and Ser87) located within the α3 helix of the DBD. Notably, DBD-deficient mutants of AjIRF2/2a retained the ability to suppress AjIFN promoter activity following poly I:C stimulation, indicating the involvement of ISRE-independent mechanisms. Further studies revealed AjIRF2/2a facilitate the ubiquitination-dependent degradation of AjSTAT1a, thereby dampening IFN signaling output. Taken together, these findings establish AjIRF2 and AjIRF2a as negative regulators of antiviral innate immunity in Japanese eel, acting through both direct transcriptional interference and post-translational modulation of key signaling components to constrain IFN responses.