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

Abstract

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.

Novel Findings in Teleost TRAF4, a Protein Acts as an Enhancer in TRIF and TRAF6 Mediated Antiviral and Inflammatory Signaling

“…These results suggest that the tissue expression patterns of TRAF4 in teleosts may vary from different species. In addition, the expression level of Lc-TRAF4 was upregulated by poly I:C, LPS, PGN, and P. plecoglossicida stimulations in gill, intestine, spleen, head kidney, and blood, which were similar to studies of other teleosts that TRAF4 in Chinese tongue sole could be induced under Vibrio harveyi infection (21), and grouper TRAF4 could be activated in response to Cryptocaryon irritans infection (19). It is thus speculated that TRAF4 functions in response to bacterial and viral infections in teleosts.…”

“…Tissue expression analysis showed that Lc - TRAF4 was widely expressed in different tissues/organs of large yellow croaker, with the highest expression level in the brain and the lowest expression level in the heart. The expression analysis of TRAF4 in other teleosts showed that the expression level of TRAF4 was the highest in the gill of Chinese tongue sole ( 21 ) and skin of grouper ( 19 ), respectively. These results suggest that the tissue expression patterns of TRAF4 in teleosts may vary from different species.…”

“…Compared to some important progress in mammals, little is known about the function of TRAF4 in teleosts. In recent years, orthologs of TRAF4 have been cloned and identified in teleosts, including Epinephelus coioides ( 19 ), Danio rerio ( 20 ), and Cynoglossus semilaevis ( 21 ). The expression level of TRAF4 was significantly upregulated under infection of Cryptocaryon irritans and red-spotted grouper nervous necrosis virus (RGNNV) in grouper ( E. coioides ).…”

“…The expression level of TRAF4 was significantly upregulated under infection of Cryptocaryon irritans and red-spotted grouper nervous necrosis virus (RGNNV) in grouper ( E. coioides ). In addition, overexpression of EcTRAF4 decreased the expression of IFN-related molecules and proinflammatory factors ( 19 ). Another report in C. semilaevis also revealed that the mRNA expression level of TRAF4 was significantly up-regulated in response to Vibrio harveyi infection ( 21 ).…”

Genome‐wide identification of the traf gene family in yellow catfish (Pelteobagrus fulvidraco) and analysis of their expression in response to bacterial challenge

“…Comparing the results of this study with those of previous studies showed that there seems to be no fixed pattern of the number zinc finger domains in the traf family in teleost fishes. For example, traf1 of grass carp (Xu et al ., 2008), traf2 of black carp (Chen et al ., 2017), traf5 of the orange‐spotted grouper (Yang et al ., 2019) and large yellow croaker traf3 and traf6 (Kang et al ., 2018) have one zinc finger domain; whereas blunt snout bream traf3 has two zinc fingers (Lv et al ., 2020), red‐spotted grouper traf4 has three zinc finger domains (Wu et al ., 2021) and traf6 of Chinese tongue sole has two zinc fingers (Li et al ., 2020). In yellow catfish, traf3 , traf4 and traf5 have two zinc finger domains, traf7 has no zinc finger domain and the rest of the proteins have one zinc finger domain.…”