International Journal of Molecular Sciences Article Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication Siting Wu 1, Mengshi Sun 1, Xin Zhang 1, Jiaming Liao 1, Mengke Liu 1, Qiwei Qin 1,2,* and Jingguang Wei 1,* 1 Guangdong Laboratory for Lingnan Modern Agriculture, Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China; [email protected] (S.W.); [email protected] (M.S.); [email protected] (X.Z.); [email protected] (J.L.); [email protected] (M.L.) 2 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, China * Correspondence: [email protected] (Q.Q.); [email protected] (J.W.) 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 Citation: Wu, S.; Sun, M.; Zhang, X.; expression in GS cells was significantly upregulated after stimulation with red-spotted grouper Liao, J.; Liu, M.; Qin, Q.; Wei, J. nervous necrosis virus (RGNNV). EcTRAF4 protein was distributed in the cytoplasm of GS cells. Grouper TRAF4, a Novel, Overexpressed EcTRAF4 promoted RGNNV replication during viral infection in vitro. Yeast two- CP-Interacting Protein That Promotes hybrid and coimmunoprecipitation assays showed that EcTRAF4 interacted with the coat protein Red-Spotted Grouper Nervous (CP) of RGNNV. EcTRAF4 inhibited the activation of IFN3, IFN-stimulated response element (ISRE), Necrosis Virus Replication. Int. J. Mol. Sci. 2021, 22, 6136. https://doi.org/ and nuclear factor-κB (NF-κB). Overexpressed EcTRAF4 also reduced the expression of interferon 10.3390/ijms22116136 (IFN)-related molecules and pro-inflammatory factors. Together, these results demonstrate that EcTRAF4 plays crucial roles in RGNNV infection. Academic Editors: Daniela Bosisio and Valentina Salvi Keywords: Epinephelus coioides; TRAF4; RGNNV; cellular localization; viral replication Received: 25 April 2021 Accepted: 3 June 2021 Published: 7 June 2021 1. Introduction Grouper, Epinephelus spp., is one of the most important marine aquaculture fish species Publisher’s Note: MDPI stays neutral in China [1]. The orange-spotted grouper, Epinephelus coioides, is a popular marine fish with regard to jurisdictional claims in cultured in Southeast Asia and China. However, for many years, outbreaks of infectious published maps and institutional affil- bacterial and viral diseases have seriously affected the grouper aquaculture industry, caus- iations. ing large economic losses [2,3]. Larval and juvenile grouper are susceptible to fatal epidemic outbreaks of diseases caused by infections with Betanodavirus or nervous necrosis viruses (NNVs) and iridoviruses [2–4]. NNVs are some of the most destructive viruses of cultured marine fishes throughout the world [5–7]. Their genomes contain two single-stranded Copyright: © 2021 by the authors. RNAs and RNA2, and RNA1 encodes the RNA-dependent RNA polymerase (RdRp) while Licensee MDPI, Basel, Switzerland. RNA2 encodes the coat protein (CP), respectively [8]. RNA1 is longer than RNA2. NNVs This article is an open access article have been classified into four primary genotypes: striped jacked NNV, red-spotted grouper distributed under the terms and NNV (RGNNV), barfin flounder NNV, and tiger puffer NNV [5]. An effective method for conditions of the Creative Commons preventing grouper virus disease is urgently required, and improving the immunity of the Attribution (CC BY) license (https:// grouper is the most promising approach to the prevention and treatment of viral infections. creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 6136. https://doi.org/10.3390/ijms22116136 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 15 Int. J. Mol. Sci. 2021, 22, 6136 effective method for preventing grouper virus disease is urgently required, and improv-2 of 14 ing the immunity of the grouper is the most promising approach to the prevention and treatment of viral infections. Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are considered the Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are considered the central signal transducers of some signaling pathways and play important roles in some central signal transducers of some signaling pathways and play important roles in some bi- biological processes such as immune regulation, inflammatory response, and apoptosis ological processes such as immune regulation, inflammatory response, and apoptosis [9,10]. [9,10]. Seven members of the TRAF family have been identified, designated TRAF1–7 [11]. Seven members of the TRAF family have been identified, designated TRAF1–7 [11]. Most MostTRAFs TRAFs (except (except TRAF7) TRAF7) contain contain a TRAF a domainTRAF domain in the C-terminalin the C-terminal region andregion a C-terminal and a C- terminalβ-sandwich β-sandwich domain (TRAF-C domain (TRAF-C or MATH or domain) MATH [12 do,13main)]. TRAFs [12,13]. also TRAFs contain also an N-terminalcontain an N-terminalRING finger RING domain finger followed domain by followed multiple by zinc mu fingerltiple zinc motifs finger [12, 14motifs]. They [12,14]. determine They de- the termineE3 ligase the activity E3 ligase of the activity TRAFs of and the areTRAFs crucial and for are the crucial activation for the of activation downstream of down- signal- streaming cascades signaling [15]. cascades TRAF4 [15]. was firstTRAF4 cloned was fromfirst cloned breast from cancer-derived breast cancer-derived metastatic lymph meta- staticnodes lymph [16,17 nodes]. TRAF4 [16,17]. contains TRAF4 a contains nuclear localizationa nuclear localization signal (NLS), signal which (NLS), is which a unique is a uniquemember member of the TRAF of the family TRAF [ 17family,18]. TRAF4[17,18]. has TRAF4 a RING has domain a RING and domain an E3 and ubiquitin an E3 ligaseubiq- uitindomain, ligase so domain, it can mediate so it can activation mediate activation of the target of the proteins target ofproteins TAK1 of and TAK1 AKT1 and and AKT1 the andK63-linked the K63-linked ubiquitination ubiquitination [19,20]. [19,20]. TRAF4 TRAF4 interacts interacts with thewith deubiquitinase the deubiquitinase USP10 USP10 and andblocks blocks the accessthe access of tumor of tumor protein protein P53 P53 (TP53) (TP53) to USP10, to USP10, destabilizing destabilizing TP53 TP53 [16 ].[16]. Unlike Un- likeother other proteins proteins of the of TRAFthe TRAF family, family, TRAF4-deficient TRAF4-deficient mice mice display display impaired impaired neural neural tube tubeclosures closures and and tracheal tracheal ring ring disruptions disruptions [21 [2].1]. TRAF4 TRAF4 can can also also interact interact weaklyweakly withwith the humanhuman p75 p75 neurotrophin neurotrophin receptor receptor (p75-NGFR), (p75-NGFR), a a member member of of the TNF-R present in the nervousnervous system, system, and and with with a lymphotoxin-beta a lymphotoxin-beta receptor receptor (LTp-R) (LTp-R) [22]. [22 TRAF4]. TRAF4 increases increases NF- κNF-B activationκB activation through through glucocorticoid-induced glucocorticoid-induced TNF-R TNF-R (GITR). (GITR). This Thiseffect effect is mediated is mediated by a TRAF-bindingby a TRAF-binding site located site located in the in cytoplasmic the cytoplasmic domain domain of GITR of GITRand is and inhibited is inhibited by cyto- by plasmiccytoplasmic protein protein A20 A20[23]. [23]. ComparedCompared with with other other teleost teleost TRAF TRAF proteins, proteins, little little is isknown known of teleost of teleost TRAF4. TRAF4. To ex- To amineexamine the the roles roles of TRAF4 of TRAF4 in the in theinnate innate immunity immunity of teleosts, of teleosts, the theTRAF4 TRAF4 homologue homologue of the of orange-spottedthe orange-spotted grouper grouper (EcTRAF4)(EcTRAF4) was wascharacterized characterized in the in present the present study. study. Next, Next, the theex- pressionexpression profiles profiles of ofEcTRAF4 EcTRAF4 were were analyzed analyzed in in the the tissues tissues of of healthy healthy fish fish and and in in GS cells afterafter viral infection. Finally,Finally, thethe effects effects of of overexpressed overexpressed EcTRAF4 EcTRAF4 on on RGNNV RGNNV proliferation prolifera- tionwere were investigated. investigated. 2.2. Results Results 2.1. Identification and Sequence Analysis of EcTRAF4 2.1. Identification and Sequence Analysis of EcTRAF4 EcTRAF4 consisted of 1413 nucleotides encoding a 471-amino-acid protein, with a EcTRAF4 consisted of 1413 nucleotides encoding a 471-amino-acid protein, with a predicted molecular mass of 54.27 kDa. No signal peptide or transmembrane helices predicted molecular mass of 54.27 kDa. No signal peptide or transmembrane