Grouper TRAF3 inhibits nodavirus infection by regulating the STING-mediated antiviral signaling pathway.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are major signal transducers for the TNF and interleukin-1/Toll-like receptor superfamilies that transduce signals from various immune receptors. To investigate the interaction of TRAF3 and other proteins in signaling pathways and to identify its antiviral function in teleosts, we cloned and characterized a TRAF3 homolog from orange-spotted grouper (Epinephelus coioides) (EcTRAF3). The open reading frame of EcTRAF3 consists of 1767 base pairs encoding a 588 amino acid protein, and the predicted molecular mass is 66.71 kDa EcTRAF3 shares 99.83% identity with TRAF3 of Epinephelus lanceolatus. Expression analysis revealed that EcTRAF3 was broadly distributed in examined tissues and was up-regulated under polyinosinic-polycytidylic acid and red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vivo. EcTRAF3 was identified as a cytosolic protein based on fluorescence microscopy analysis. Overexpression of EcTRAF3 inhibited RGNNV replication in grouper spleen cells, and it interacted with the coat protein of RGNNV. Overexpression of EcTRAF3 also induced the activation of interferon ß (IFN-ß), IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). EcTRAF3 co-transfected with Stimulator of Interferon Genes (STING) of grouper (EcSTING) induced a significantly higher level of IFN-ß promoter activity. Moreover, EcTRAF3 interacted with EcSTING, implying that EcTRAF3 may function as an enhancer in EcSTING-mediated signaling. Taken together, our results suggest that EcTRAF3 negatively regulates the RGNNV-induced cellular antiviral response and plays an important role in the immune response system of fish.

Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication.

Tumor necrosis factor receptor-associated factors (TRAFs) play important roles in the biological processes of immune regulation, the inflammatory response, and apoptosis. TRAF4 belongs to the TRAF family and plays a major role in many biological processes. Compared with other TRAF proteins, the functions of TRAF4 in teleosts have been largely unknown. In the present study, the TRAF4 homologue (EcTRAF4) of the orange-spotted grouper was characterized. EcTRAF4 consisted of 1413 bp encoding a 471-amino-acid protein, and the predicted molecular mass was 54.27 kDa. EcTRAF4 shares 99.79% of its identity with TRAF4 of the giant grouper (E. lanceolatus). EcTRAF4 transcripts were ubiquitously and differentially expressed in all the examined tissues. EcTRAF4 expression in GS cells was significantly upregulated after stimulation with red-spotted grouper nervous necrosis virus (RGNNV). EcTRAF4 protein was distributed in the cytoplasm of GS cells. Overexpressed EcTRAF4 promoted RGNNV replication during viral infection in vitro. Yeast two-hybrid and coimmunoprecipitation assays showed that EcTRAF4 interacted with the coat protein (CP) of RGNNV. EcTRAF4 inhibited the activation of IFN3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). Overexpressed EcTRAF4 also reduced the expression of interferon (IFN)-related molecules and pro-inflammatory factors. Together, these results demonstrate that EcTRAF4 plays crucial roles in RGNNV infection.

Grouper TRAF5 exerts negative regulation on antiviral immune response against iridovirus.

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an intracellular protein that binds to the cytoplasmic portion of tumor necrosis factor receptors and mediates the activation of downstream nuclear factor-kappa B (NF-κB), interferon regulatory factor 3, and mitogen activated protein kinase signaling pathways. Compared with other TRAF proteins, TRAF5 is largely unknown in teleosts. In the present study, a TRAF5 homologue (HgTRAF5) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) was cloned and characterized. The open reading frame of HgTRAF5 consists of 1743 nucleotides encoding a 581 amino acid protein with a predicted molecular mass of 64.90 kDa. Similar to its mammalian counterpart, HgTRAF5 contains an N-terminal RING finger domain, a zinc finger domain, and a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. HgTRAF5 shares 99.83% identity with giant grouper (Epinephelus lanceolatus) TRAF5. Quantitative real-time PCR analysis indicated that HgTRAF5 mRNA was broadly expressed in all examined tissues. The expression of HgTRAF5 increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that the full-length HgTRAF5 protein mainly distributed in the cytoplasm. HgTRAF5 overexpression also promoted SGIV replication during viral infection in vitro. HgTRAF5 significantly promoted the activities of interferon-ß, interferon-sensitive response element, and NF-κB. Taken together, these results are important for a better understanding of the function of TRAF5 in fish and reveal its involvement in the host response to immune challenge by SGIV.

Fish RIP1 Mediates Innate Antiviral Immune Responses Induced by SGIV and RGNNV Infection.

Receptor interacting protein 1 (RIP1) is an essential sensor of cellular stress, which may respond to apoptosis or cell survival and participate in antiviral pathways. To investigate the roles of fish RIP1 in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, a RIP1 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP1) was cloned and characterized. EcRIP1 encoded a 679 amino acid protein that shares 83.28% identity with that of Perca flavescens and contained a homologous N-terminal kinase (S-TKc) domain, a RIP isotype interaction motif (RHIM), and a C-terminal domain (DD). EcRIP1 was predominantly detected in immune tissues, and its expression was induced by RGNNV or SGIV infection in vitro. Subcellular localization showed that EcRIP1 was distributed in the cytoplasm with point-like uniform and dot-like aggregation forms. Overexpression of EcRIP1 inhibited SGIV and RGNNV replication and positively regulated the expression levels of interferon (IFN) and IFN-stimulated genes and pro-inflammatory factors. EcRIP1 may interact with grouper tumor necrosis factor receptor type 1-associated DEATH domain protein (EcTRADD) to promote SGIV-induced apoptosis, and interact with grouper Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-ß (EcTRIF) and participate in Myeloid Differentiation Factor 88 (MyD88)-independent toll-like receptor (TLR) signaling. EcRIP1 may also interact with grouper tumor necrosis factor receptor-associated factors (TRAFs) as intracellular linker proteins and mediate the signaling of various downstream signaling pathways, including NF-κB and IFN. These results suggest that EcRIP1 may inhibit SGIV and RGNNV infection by regulating apoptosis and various signaling molecules. Our study offers new insights into the regulatory mechanism of RIP1-related signaling, and provides a novel perspective on fish diseases mediated by RIP1.

Fish TRAF2 promotes innate immune response to RGNNV infection.

Tumour necrosis factor receptor-associated factors (TRAFs) are key regulatory proteins in the NF-κB signaling pathways. TRAF2 participates in the activation of both canonical and non-canonical NF-κB 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.