Characterization of tumor necrosis factor receptor-associated factor 2 (TRAF2) in red-spotted grouper (Epinephelus akaara): In vivo and in vitro investigation of its role in the regulation of antiviral immunity and cell death

Abstract

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a key adaptor molecule in tumor necrosis factor receptor signaling complexes, facilitating downstream immune-related signaling cascades. This study aimed to elucidate its function in teleost fish by characterizing the TRAF2 homolog of the red-spotted grouper (Epinephelus akaara, EaTraf2). The open reading frame of EaTraf2 encodes a putative protein of 520 amino acids, containing several characteristic domains of TRAF2. These structural features of EaTraf2 are conserved across diverse organisms, with a relatively higher sequence identity to TRAF2 orthologs from other bony fish. Transcriptional analysis demonstrated that EaTraf2 was ubiquitously expressed in all examined tissues, with the highest level observed in blood. Upon immune challenge, EaTraf2 expression significantly increased in the early stages of stimulation in both blood and spleen. Subcellular localization analysis revealed that EaTraf2 is predominantly localized in the cytoplasm. Overexpression of EaTraf2 in fathead minnow (FHM) cells resulted in elevated levels of interferon and inflammation-associated genes following viral hemorrhagic septicemia virus (VHSV) infection, along with reduced viral gene expression. This provided compelling evidence that EaTraf2 possesses antiviral properties. Furthermore, EaTraf2 demonstrated the ability to promote cell death induced by oxidative stress. Additionally, luciferase reporter assays revealed that EaTraf2 activates the NF-κB signaling pathway upon poly(I:C) stimulation and the Jun N-terminal kinase (JNK) signaling pathway in response to H₂O₂ treatment. Overall, our study elucidated the role of EaTraf2 in regulating innate immune responses and mediating stress-induced cell death. These findings enhance our understanding of TRAF2 in fish and may contribute to improved health management strategies in finfish aquaculture.