Xu Dan, Song Lang, Wang Hao, Xu Xiaoyan, Wang Tu, Lu Liqun
Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, PR China.
Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, PR China.
Fish Shellfish Immunol. 2015 Jun;44(2):515-24. doi: 10.1016/j.fsi.2015.03.010. Epub 2015 Mar 14.
Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. To better understand the molecular responses to GCRV infection, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization tandem mass spectroscopy were performed to investigate altered proteins in C. idella kidney (CIK) cells. Differentially expressed proteins in mock infected CIK cells and GCRV-infected CIK cells were compared. Twenty-three differentially expressed spots were identified (22 upregulated spots and 1 downregulated spot), which included cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism-associated proteins and ubiquitin proteasome pathway-associated proteins. Moreover, 10 of the corresponding genes of the differentially expressed proteins were quantified by real-time reverse transcription polymerase chain reaction to examine their transcriptional profiles. The T cell internal antigen 1 (TIA1) and Ras-GTPase-activating SH3-domain-binding protein1 (G3BP1) of the cellular stress granule pathway from grass carp C. idella (designated as CiTIA1 and CiG3BP1) were upregulated and downregulated during GCRV infection, respectively. The full-length cDNA of CiTIA1 was 2753 bp, with an open reading frame (ORF) of 1155bp, which encodes a putative 385-amino acid protein. The 2271 bp full-length cDNA of CiG3BP1 comprised an ORF of 1455 bp that encodes a putative 485-amino acid protein. Phylogenetic analysis revealed that the complete ORFs of CiTIA1 and CiG3BP1 were very similar to zebrafish and well-characterized mammalian homologs. The expressions of the cellular proteins CiTIA1 and CiG3BP1 in response to GCRV were validated by western blotting, which indicated that the GCRV should unlink TIA1 aggregation and stress granule formation. This study provides useful information on the proteomic and cellular stress granule pathway's responses to GCRV infection, which adds to our understanding of viral pathogenesis.
草鱼呼肠孤病毒（GCRV）引起的草鱼（Ctenopharyngodon idella）出血病，正成为草鱼养殖中的一个严重问题。为了更好地了解对GCRV感染的分子反应，进行了二维电泳（2-DE）和基质辅助激光解吸/电离串联质谱分析，以研究草鱼肾脏（CIK）细胞中变化的蛋白质。比较了模拟感染的CIK细胞和GCRV感染的CIK细胞中差异表达的蛋白质。鉴定出23个差异表达的斑点（22个上调斑点和1个下调斑点），其中包括细胞骨架蛋白、大分子生物合成相关蛋白、应激反应蛋白、信号转导蛋白、能量代谢相关蛋白和泛素蛋白酶体途径相关蛋白。此外，通过实时逆转录聚合酶链反应对差异表达蛋白质的10个相应基因进行定量，以检查它们的转录谱。草鱼C. idella细胞应激颗粒途径中的T细胞内抗原1（TIA1）和Ras-GTP酶激活SH3结构域结合蛋白1（G3BP1）（分别命名为CiTIA1和CiG3BP1）在GCRV感染期间分别上调和下调。CiTIA1的全长cDNA为2753 bp，开放阅读框（ORF）为1155 bp，编码一个推定的385个氨基酸的蛋白质。CiG3BP1的2271 bp全长cDNA包含一个1455 bp的ORF，编码一个推定的485个氨基酸的蛋白质。系统发育分析表明，CiTIA1和CiG3BP1的完整ORF与斑马鱼以及特征明确的哺乳动物同源物非常相似。通过蛋白质免疫印迹法验证了细胞蛋白CiTIA1和CiG3BP1对GCRV的反应，这表明GCRV应该解除TIA1聚集和应激颗粒形成的联系。本研究提供了关于蛋白质组学和细胞应激颗粒途径对GCRV感染反应的有用信息，增进了我们对病毒发病机制的理解。
