Department of Life Science, National Taiwan University, Taipei, Taiwan.
Department of Life Science, National Taiwan University, Taipei, Taiwan
J Virol. 2015 Jan;89(1):61-70. doi: 10.1128/JVI.02523-14. Epub 2014 Oct 15.
Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses of Nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were investigated as NNV receptor protein candidates. We cloned and sequenced the genes for GHSC70 and GVDAC2 and expressed them in Escherichia coli for antiserum preparation. Knockdown of the expression of GHSC70 and GVDAC2 genes with specific short interfering RNAs (siRNAs) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. By performing an immunoprecipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the presence of the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 levels after 1 h of adsorption to GF-1 cells; the level was significantly lower in the cells pretreated with the GHSC70 antiserum than in nontreated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functioning as an NNV receptor or coreceptor protein.
Fish nodavirus has caused mass mortality of more than 40 fish species worldwide and resulted in huge economic losses in the past 20 years. Among the four genotypes of fish nodaviruses, the red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating that NNV-specific receptor(s) may exist on the GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be an NNV receptor (or coreceptor) candidate through VOBPA and provided several lines of evidence demonstrating that GHSC70 protein has a role in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.
神经坏死病毒 (NNV) 是一种严重危害养殖海水鱼的病原体，已影响 40 多种鱼类。NNV 属于弹状病毒科的甲病毒，是一种无包膜的二十面体颗粒，含有 2 条单链正链 RNA。迄今为止，关于 NNV 进入宿主细胞的知识仍然有限，并且尚未发表任何 NNV 特异性受体蛋白。在病毒覆盖蛋白结合测定 (VOPBA) 中，使用石斑鱼鳍细胞系 GF-1 和纯化的 NNV 衣壳蛋白，研究了石斑鱼热休克同源蛋白 70 (GHSC70) 和石斑鱼电压依赖性阴离子选择性通道蛋白 2 (GVDAC2) 是否为 NNV 受体蛋白候选物。我们克隆并测序了 GHSC70 和 GVDAC2 的基因，并在大肠杆菌中表达以制备抗血清。用特异性小干扰 RNA (siRNA) 敲低 GHSC70 和 GVDAC2 基因的表达，显著下调了 NNV 感染的 GF-1 细胞中的病毒 RNA 表达。通过免疫沉淀测定，我们证实 GHSC70 与 NNV 衣壳蛋白相互作用，而 VDAC2 则没有。免疫荧光染色和流式细胞术分析显示 GHSC70 蛋白存在于细胞表面。在阻断试验后，我们检测了 NNV RNA2 在吸附到 GF-1 细胞 1 小时后的水平；用 GHSC70 抗血清预处理的细胞中的水平明显低于未处理的细胞。因此，我们认为 GHSC70 参与了 GF-1 细胞的 NNV 进入，可能作为 NNV 的受体或辅助受体蛋白发挥作用。
鱼类诺达病毒在过去 20 年中已导致全球 40 多种鱼类大量死亡，造成巨大的经济损失。在鱼类诺达病毒的四个基因型中，红鳍东方鲀神经坏死病毒 (RGNNV) 基因型表现出最广泛的宿主范围。在我们之前的研究中，我们在 GF-1 细胞中开发了针对石斑鱼 NNV 的具有高中和效率的单克隆抗体，表明 NNV 特异性受体 (s) 可能存在于 GF-1 细胞膜上。然而，尚未发表任何 NNV 受体蛋白。在这项研究中，我们通过 VOBPA 发现 GHSC70 是 NNV 的受体 (或辅助受体) 候选物，并提供了几条证据表明 GHSC70 蛋白在 NNV 进入 GF-1 细胞的步骤中发挥作用。据我们所知，这是首次鉴定出石斑鱼 HSC70 及其在 NNV 进入 GF-1 细胞中的作用。
