State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China.
State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China.
Dev Comp Immunol. 2019 Jul;96:68-77. doi: 10.1016/j.dci.2019.03.001. Epub 2019 Mar 7.
NOD-like receptor (NLR) family member X1 (NLRX1) of human localizes on mitochondria and serves as a negative regulator of antiviral signaling. However, the function of NLRX1 in teleost fish still remains elusive. To explore its role in the innate immunity of teleost fish, NLRX1 homologue has been cloned and characterized from black carp (Mylopharyngodon piceus). Black carp NLRX1 (bcNLRX1) consists of 1008 amino acids, which includes a N-terminal mitochondrial targeting sequence, a central NACHT domain and a C-terminal leucine-rich repeat (LRR) domain. bcNLRX1 was identified as a cytosolic protein locating on mitochondria through immunofluorescence (IF) staining. The overlapped subcellular distribution of bcNLRX1 and black carp MAVS (bcMAVS) was detected in IF staining, and the direct interaction between these two molecules in vitro was identified through co-immunoprecipitation assay. When co-expressed with bcMAVS, bcNLRX1 fiercely reduced bcMAVS-mediated IFN induction in reporter assay. Accordingly, the antiviral activity of bcMAVS against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV) was forcefully repressed by bcNLRX1 in plaque assay. Mutagenic analyses further revealed that the NACHT domain of bcNLRX1 was essential for it to interact with bcMAVS and to suppress bcMAVS-mediated antiviral signaling. Taken together, our data support the conclusion that bcNLRX1 negatively regulates bcMAVS-mediated antiviral signaling through its NACHT domain during host innate immune activation.
核苷酸结合寡聚化结构域样受体（NLR）家族成员 X1（NLRX1）位于线粒体上，作为抗病毒信号的负调控因子。然而，NLRX1 在硬骨鱼类中的功能仍然难以捉摸。为了探索其在硬骨鱼类先天免疫中的作用，从草鱼（Mylopharyngodon piceus）中克隆并鉴定了 NLRX1 同源物。草鱼 NLRX1（bcNLRX1）由 1008 个氨基酸组成，包括一个 N 端线粒体靶向序列、一个中央 NACHT 结构域和一个 C 端富含亮氨酸重复（LRR）结构域。通过免疫荧光（IF）染色鉴定 bcNLRX1 为定位于线粒体的细胞质蛋白。IF 染色检测到 bcNLRX1 和草鱼 MAVS（bcMAVS）的重叠亚细胞分布，并且通过共免疫沉淀实验鉴定了这两种分子在体外的直接相互作用。当与 bcMAVS 共表达时，bcNLRX1 在报告基因检测中强烈降低了 bcMAVS 介导的 IFN 诱导。相应地，bcNLRX1 在斑块测定中强烈抑制了 bcMAVS 对草鱼呼肠孤病毒（GCRV）和鲤鱼出血性败血症病毒（SVCV）的抗病毒活性。突变分析进一步表明，bcNLRX1 的 NACHT 结构域对于其与 bcMAVS 相互作用和抑制 bcMAVS 介导的抗病毒信号转导是必不可少的。总之，我们的数据支持这样的结论，即 bcNLRX1 通过其 NACHT 结构域在宿主先天免疫激活过程中负调控 bcMAVS 介导的抗病毒信号转导。
