Wu Siting, Sun Mengshi, Zhang Luhao, Kang Shaozhu, Liao Jiaming, Zhu Zheng, Chen Hong, Xu Zhuqing, Xu Linting, Zhang Xin, Wei Jingguang, Qin Qiwei
University Joint Laboratory of Guangdong Province, Hong Kong and Macao Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, PR China.
University Joint Laboratory of Guangdong Province, Hong Kong and Macao Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, PR China.
Fish Shellfish Immunol. 2022 Apr;123:172-181. doi: 10.1016/j.fsi.2022.03.001. Epub 2022 Mar 8.
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 β (IFN-β), 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-β 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.
肿瘤坏死因子（TNF）受体相关因子（TRAFs）是TNF和白细胞介素-1/Toll样受体超家族的主要信号转导分子，可转导来自各种免疫受体的信号。为了研究TRAF3与信号通路中其他蛋白质的相互作用，并确定其在硬骨鱼中的抗病毒功能，我们克隆并鉴定了来自点带石斑鱼（Epinephelus coioides）的TRAF3同源物（EcTRAF3）。EcTRAF3的开放阅读框由1767个碱基对组成，编码一个588个氨基酸的蛋白质，预测分子量为66.71 kDa。EcTRAF3与鞍带石斑鱼的TRAF3具有99.83%的同一性。表达分析表明，EcTRAF3广泛分布于所检测的组织中，并且在体内经聚肌苷酸-聚胞苷酸和红斑石斑鱼神经坏死病毒（RGNNV）刺激后上调。基于荧光显微镜分析，EcTRAF3被鉴定为一种胞质蛋白。EcTRAF3的过表达抑制了RGNNV在石斑鱼脾脏细胞中的复制，并且它与RGNNV的衣壳蛋白相互作用。EcTRAF3的过表达还诱导了干扰素β（IFN-β）、干扰素刺激反应元件（ISRE）和核因子κB（NF-κB）的激活。与石斑鱼干扰素基因刺激物（STING）（EcSTING）共转染的EcTRAF3诱导了显著更高水平的IFN-β启动子活性。此外，EcTRAF3与EcSTING相互作用，这意味着EcTRAF3可能在EcSTING介导的信号传导中起增强子的作用。综上所述，我们的结果表明，EcTRAF3负调控RGNNV诱导的细胞抗病毒反应，并在鱼类免疫反应系统中发挥重要作用。
