The E3 ligase RNF5 facilitates viral replication by suppressing innate immune responses in grouper

Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) are major pathogens that cause high mortality and substantial economic losses in grouper aquaculture. The role of ring finger protein 5 (RNF5), an E3 ubiquitin ligase, in the antiviral innate immunity of teleosts remains poorly understood. In this study, a homolog of the RNF5 gene was identified and functionally characterized from Epinephelus coioides, which was designated EcRNF5. The open reading frame (ORF) of EcRNF5 is 645 nucleotides in length, encoding a 214-amino acid protein that contains a characteristic RING domain. Phylogenetic analysis revealed that EcRNF5 shares the closest evolutionary relationship with its ortholog in Epinephelus fuscoguttatus. EcRNF5 was ubiquitously expressed in all examined tissues. Furthermore, its transcription level was significantly upregulated in grouper spleen (GS) cells following challenge with SGIV or RGNNV. Overexpression of EcRNF5 enhanced SGIV and RGNNV replication in vitro. Concurrently, it suppressed the expression of interferon-related genes and pro-inflammatory cytokines. The promoter activities of interferon 3 (IFN3), interferon-stimulated response element (ISRE), and nuclear factor kappa B (NF-κB) were also inhibited. In addition, EcRNF5 attenuated IFN3 promoter activation induced by the key signaling molecules: EcSTING, EcTBK1, EcIRF3, and EcIRF7. Co-immunoprecipitation (Co-IP) assays confirmed physical interactions between EcRNF5 and these four innate immune signaling molecules. Collectively, these findings provide novel insights into the functional role of RNF5 in fish-virus interactions and shed light on the molecular mechanisms underlying SGIV and RGNNV pathogenicity in grouper.