College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.
College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.
Fish Shellfish Immunol. 2019 May;88:391-402. doi: 10.1016/j.fsi.2019.03.004. Epub 2019 Mar 7.
Tripartite motif (TRIM) proteins have been demonstrated to exhibit critical functions in multiple cellular processes, including development, carcinogenesis, and programmed cell death, and are also widely recognized to be important antiviral restriction factors or modulators of immune and inflammatory signaling pathways. However, in teleosts, additional TRIM members have been identified and their functions remain largely unknown. Here, a novel finTRIM gene from orange spotted grouper (EcfinTRIM82) was cloned and characterized. Sequence analysis indicated that EcfinTRIM82 encoded a 575 amino acid peptide which shared 94% and 82% identity with Asian sea bass (Lates calcarifer), and zebrafish (Danio rerio) finTRIM82, respectively. EcfinTRIM82 contained three conserved domains, including a RING, B-Box, and SPRY domain. Using fluorescence microscopy, we found that green fluorescence aggregates were observed in the cytoplasm of EcfinTRIM82-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcfinTRIM82 transcription was significantly upregulated in Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infected GS cells. This suggests that EcfinTRIM82 might be involved in fish virus infection. The in vitro overexpression of EcfinTRIM82 in GS cells significantly enhanced the replication of SGIV and RGNNV, evidenced by increased expression of viral genes, including the SGIV major capsid protein (MCP), VP19, ICP-18, RGNNV coat protein (CP), and RNA-dependent RNA polymerase (RdRp). Furthermore, the ectopic expression of EcfinTRIM82 significantly decreased the expression of interferon (IFN)-related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), ISG56, IFP35, and myxovirus resistance gene (MXI), suggesting that EcfinTRIM82 regulated viral replication via the negative regulation of the host IFN response. In addition, EcfinTRIM82 overexpression substantially decreased the level of proinflammatory cytokine transcription. Furthermore, the ectopic expression of EcfinTRIM82 significantly weakened the melanoma differentiation-associated protein 5 (MDA5), mediator of IRF3 activation (MITA) and mitochondrial antiviral-signaling (MAVS) protein-induced IFN response by detecting the transcription of interferon related cytokines and the promoter activity of IFN. Together, our results demonstrate that finTRIM82 negatively regulates the innate antiviral immune response against grouper virus infection.
三结构域蛋白 (TRIM) 已被证明在多种细胞过程中发挥关键作用，包括发育、致癌和程序性细胞死亡，并且还被广泛认为是重要的抗病毒限制因子或免疫和炎症信号通路的调节剂。然而，在硬骨鱼类中，已经鉴定出更多的 TRIM 成员，但其功能仍知之甚少。在这里，我们从橙色斑点石斑鱼中克隆并表征了一种新型 finTRIM 基因（EcfinTRIM82）。序列分析表明，EcfinTRIM82 编码一个 575 个氨基酸的肽，与亚洲海鲈（Lates calcarifer）和斑马鱼（Danio rerio）finTRIM82 分别具有 94%和 82%的同一性。EcfinTRIM82 包含三个保守结构域，包括一个 RING、B-Box 和 SPRY 结构域。通过荧光显微镜，我们发现 EcfinTRIM82-EGFP 转染石斑鱼脾脏（GS）细胞的细胞质中观察到绿色荧光聚集。随着感染的进行，EcfinTRIM82 在新加坡石斑鱼虹彩病毒（SGIV）或红鳍东方鲀神经坏死病毒（RGNNV）感染的 GS 细胞中的转录显著上调。这表明 EcfinTRIM82 可能参与鱼类病毒感染。在 GS 细胞中体外过表达 EcfinTRIM82 显著增强了 SGIV 和 RGNNV 的复制，这表现在病毒基因的表达增加，包括 SGIV 主要衣壳蛋白（MCP）、VP19、ICP-18、RGNNV 衣壳蛋白（CP）和 RNA 依赖性 RNA 聚合酶（RdRp）。此外，EcfinTRIM82 的异位表达显著降低了干扰素（IFN）相关信号分子的表达，包括干扰素调节因子 3（IRF3）、IRF7、干扰素刺激基因 15（ISG15）、ISG56、IFP35 和抗粘液病毒基因（MXI），表明 EcfinTRIM82 通过负调控宿主 IFN 反应来调节病毒复制。此外，EcfinTRIM82 的过表达显著降低了促炎细胞因子转录水平。此外，通过检测干扰素相关细胞因子的转录和 IFN 启动子活性，EcfinTRIM82 的异位表达显著削弱了黑色素瘤分化相关蛋白 5（MDA5）、IRF3 激活介质（MITA）和线粒体抗病毒信号（MAVS）蛋白诱导的 IFN 反应。总之，我们的结果表明 finTRIM82 负调控石斑鱼病毒感染的固有抗病毒免疫反应。
