Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China; University of Chinese Academy of Sciences, Beijing, China.
Fish Shellfish Immunol. 2016 Aug;55:106-15. doi: 10.1016/j.fsi.2016.05.029. Epub 2016 May 24.
The tripartite motif (TRIM)-containing proteins have attracted particular attention to their multiple functions in different biological processes. TRIM13, a member of the TRIM family, is a RING domain-containing E3 ubiquitin ligase which plays critical roles in diverse cellular processes including cell death, cancer and antiviral immunity. In this study, a TRIM13 homolog from orange spotted grouper, Epinephelus coioides (EcTRIM13) was cloned and characterized. The full-length of EcTRIM13 cDNA encoded a polypeptide of 399 amino acids which shared 81% identity with TRIM13 homolog from large yellow croaker (Larimichthys crocea). Amino acid alignment analysis showed that EcTRIM13 contained conserved RING finger and B-box domain. Expression patterns analysis indicated that EcTRIM13 was abundant in liver, spleen, kidney, intestine and gill. Moreover, the transcript of EcTRIM13 in grouper spleen was differently regulated after injection with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C). Under fluorescence microscopy, we observed the tubular structure in wild type EcTRIM13 transfected cells, but the RING domain mutant resulted in the fluorescence distribution was changed and the bright punctate fluorescence was evenly situated throughout the cytoplasm, suggesting that the RING domain was essential for its accurate localization. Overexpression of EcTRIM13 in vitro obviously increased the replication of red spotted grouper nervous necrosis virus (RGNNV), and the enhancing effect of EcTRIM13 on virus replication was affected by the RING domain. Furthermore, the ectopic expression of EcTRIM13 not only negatively regulated the interferon promoter activity induced by interferon regulator factor (IRF) 3, IRF7, and melanoma differentiation-associated protein 5 (MDA5), but also decreased the expression of several interferon related factors. In addition, the overexpression of EcTRIM13 also differently regulated the transcription of pro-inflammatory factors. Together, our results firstly demonstrated that fish TRIM13 exerted negative regulation of antiviral response against nodavirus infection.
三结构域蛋白（TRIM）因其在多种生物学过程中的多种功能而备受关注。TRIM13 是 TRIM 家族的一员，是一种含有 RING 结构域的 E3 泛素连接酶，在包括细胞死亡、癌症和抗病毒免疫在内的多种细胞过程中发挥关键作用。在这项研究中，克隆并鉴定了橙色斑点石斑鱼（Epinephelus coioides）的 TRIM13 同源物（EcTRIM13）。EcTRIM13 的全长 cDNA 编码一个由 399 个氨基酸组成的多肽，与大黄鱼（Larimichthys crocea）的 TRIM13 同源物具有 81%的同一性。氨基酸比对分析表明，EcTRIM13 含有保守的 RING 指和 B 盒结构域。表达模式分析表明，EcTRIM13 在肝脏、脾脏、肾脏、肠道和鳃中丰富表达。此外，注射新加坡石斑鱼虹彩病毒（SGIV）或聚肌苷酸-聚胞苷酸（poly I:C）后，石斑鱼脾脏中的 EcTRIM13 转录物受到不同的调控。在荧光显微镜下，我们观察到转染野生型 EcTRIM13 的细胞中存在管状结构，但 RING 结构域突变导致荧光分布发生改变，明亮的点状荧光均匀分布在细胞质中，表明 RING 结构域对于其准确定位是必不可少的。体外过表达 EcTRIM13 明显增加了红鳍东方鲀神经坏死病毒（RGNNV）的复制，并且 EcTRIM13 对病毒复制的增强作用受 RING 结构域的影响。此外，EcTRIM13 的异位表达不仅负调控干扰素调节因子（IRF）3、IRF7 和黑色素瘤分化相关蛋白 5（MDA5）诱导的干扰素启动子活性，还降低了几种干扰素相关因子的表达。此外，EcTRIM13 的过表达也不同程度地调节了促炎因子的转录。总之，我们的研究结果首次表明鱼类 TRIM13 对神经坏死病毒感染的抗病毒反应具有负调控作用。
