Fish TRAF2 promotes innate immune response to RGNNV infection

Tumour necrosis factor receptor-associated factors (TRAFs) are key regulatory proteins in the NF-kappa B signaling pathways. TRAF2 participates in the activation of both canonical and non-canonical NF-kappa B pathways, which are crucial for cell inflammation and cell survival. To elucidate its function in teleost fish, TRAF2 homologues of yellow grouper (Epinephelus awoara) and golden pompano (Trachinotus ovatus) have been cloned and characterized in this study. The open reading frame (ORF) of grouper TRAF2 (EaTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.70 kDa. The ORF of golden pompano TRAF2 (ToTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.66 kDa EaTRAF2 and ToTRAF2 share 99.23% and 99.42% identity with orange-spotted grouper (Epinephelus coioides) TRAF2 (EcTRAF2), respectively. Quantitative real-time PCR analysis indicated that the expression of EaTRAF2 was increased in grouper spleen (GS) cells after Red-spotted grouper nervous necrosis virus (RGNNV) infection; while the expression of ToTRAF2 was decreased in golden pompano brain (TOGB) cells after RGNNV infection. Both EaTRAF2 and ToTRAF2 were identified as a cytosolic protein and suggested to be associated with vesicles scattering in the cytoplasm. Both EaTRAF2 and ToTRAF2 enhanced RGNNV replication during viral infection in vitro. Further studies showed that EaTRAF2 and ToTRAF2 overexpression decreased the expression levels of interferon associated cytokines and pro-inflammatory factors. Taken together, these results are important for better understanding of the function of TRAF2 in fish and reveal its involvement in host response to immune challenges in RGNNV.