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 Sep;92:746-755. doi: 10.1016/j.fsi.2019.06.062. Epub 2019 Jul 3.
Accumulated evidence suggests that some of the tripartite motif (TRIM) -family proteins function as critical regulators of carcinogenesis, immunity, and antiviral functions. TRIM44 is an atypical TRIM family protein that lacks the entire RING domain and has been demonstrated to play a crucial role in cancer and viral infection. To our knowledge, the role of TRIM44 in fish still remains largely unknown. Here, we cloned and characterized a novel TRIM44-like gene from orange spotted grouper (EcTRIM44L). Sequence analysis indicated that EcTRIM44L encoded a 393 amino acid peptide, which shared 81.44% and 51.02% identity with large yellow croaker (Larimichthys crocea) and zebrafish (Danio rerio), respectively. However, EcTRIM44L only exhibited 24.69% identity with the TRIM44 protein of humans (Homo sapiens). Moreover, EcTRIM44L contained two conserved domains, including a B-Box domain and a coiled-coil domain, but not a RING domain. Using fluorescence microscopy, we observed green fluorescence in the cytoplasm of the EcTRIM44L-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcTRIM44L transcription was significantly up-regulated in red-spotted grouper nervous necrosis virus (RGNNV) infection, suggesting that EcTRIM44L might be involved in fish virus infections. The in vitro overexpression of EcTRIM44L significantly enhanced RGNNV replication, as demonstrated by the accelerated cytopathic effect (CPE) progression induced by RGNNV, as well as the increased expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcTRIM44L significantly decreased the level of interferon (IFN) related signaling molecules and pro-inflammatory cytokine expression, suggesting that EcTRIM44L affected virus replication by negatively regulating the IFN response. In addition, the melanoma differentiation-associated protein 5 (MDA5) and mitochondrial antiviral-signaling protein (MAVS), but not mediator of IRF3 activation (MITA)-evoked IFN response was negatively regulated by EcTRIM44L. Together, for the first time, our results indicate that EcTRIM44L negatively regulates the interferon response against grouper RNA virus infection.
积累的证据表明，一些三联基序（TRIM）家族蛋白作为致癌作用、免疫和抗病毒功能的关键调节剂。TRIM44 是一种非典型的 TRIM 家族蛋白，缺乏整个 RING 结构域，已被证明在癌症和病毒感染中发挥关键作用。据我们所知，TRIM44 在鱼类中的作用在很大程度上仍然未知。在这里，我们从橙色斑点石斑鱼（EcTRIM44L）中克隆并鉴定了一种新型的 TRIM44 样基因。序列分析表明，EcTRIM44L 编码一个 393 个氨基酸的肽，与大黄鱼（Larimichthys crocea）和斑马鱼（Danio rerio）分别具有 81.44%和 51.02%的同一性。然而，EcTRIM44L 与人类（Homo sapiens）的 TRIM44 蛋白仅显示 24.69%的同一性。此外，EcTRIM44L 含有两个保守结构域，包括一个 B-Box 结构域和一个卷曲螺旋结构域，但没有 RING 结构域。通过荧光显微镜观察，我们观察到 EcTRIM44L-EGFP 转染石斑鱼脾脏（GS）细胞的细胞质中呈现绿色荧光。随着感染的进行，EcTRIM44L 在红鳍东方鲀神经坏死病毒（RGNNV）感染中的转录明显上调，表明 EcTRIM44L 可能参与鱼类病毒感染。体外过表达 EcTRIM44L 显著增强了 RGNNV 的复制，这表现为 RGNNV 诱导的致病变效应（CPE）进展加速，以及外壳蛋白（CP）和 RNA 依赖性 RNA 聚合酶（RdRp）的表达增加。EcTRIM44L 的过表达显著降低了干扰素（IFN）相关信号分子和促炎细胞因子表达水平，表明 EcTRIM44L 通过负调控 IFN 反应影响病毒复制。此外，黑色素瘤分化相关蛋白 5（MDA5）和线粒体抗病毒信号蛋白（MAVS），而不是干扰素反应介质激活（MITA）诱导的 IFN 反应被 EcTRIM44L 负调控。总的来说，我们的研究结果首次表明，EcTRIM44L 负调控石斑鱼 RNA 病毒感染的干扰素反应。
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