Grass carp reovirus NS26 interacts with cellular lipopolysaccharide-induced tumor necrosis factor-alpha factor, LITAF
Grass carp reovirus NS26 interacts with cellular lipopolysaccharide-induced tumor necrosis factor-alpha factor, LITAF
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草鱼呼肠孤病毒 NS26 与细胞脂多糖诱导的肿瘤坏死因子-α因子 (LITAF) 相互作用
DOI：
10.1007/s11262-016-1370-6
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发表时间：
2016-12-01
期刊：
影响因子：
1.6
通讯作者：
Lu, Liqun
中科院分区：
文献类型：
--
作者：
Lu, Jianfei;Wang, Hao;Lu, Liqun
草鱼呼肠孤病毒（Grasscarpreovirus，GCRV）的非结构蛋白NS 26由基因组第11个dsRNA片段编码，在正呼肠孤病毒中未发现其同源物。NS 26在GCRV发病机制中的作用尚不清楚。之前，草鱼LITAF/SIMPLE蛋白在酵母双杂交筛选中被鉴定为NS 26的推定结合伴侣。在这里，我们使用体内和体外蛋白质相互作用测定进一步表征了NS 26和LITAF之间的关联。弗罗姆中表达并纯化了可溶性GST-NS 26和His 6-LITAF。大肠杆菌;用瞬时转染法在CIK细胞中表达了myc标记的重组NS 26和GFP标记的重组LITAF。GST pulldown分析证明GST标记的NS 26有效地结合His 6-LITAF。免疫共沉淀试验表明，GCRV NS 26可在CIK细胞中沉淀内源性LITAF。免疫荧光双标分析显示，myc-NS 26与GFP-LITAF共定位于CIK细胞中。总之，目前的体外和体内数据证明了细胞LITAF和GCRV NS 26之间的相互作用。
The nonstructural protein NS26 of grass carp reovirus (GCRV) is encoded by the 11th genomic dsRNA segment, homolog of which is not found inorthoreoviruses. The role of NS26 in GCRV pathogenesis is still unclear. Previously, grass carp LITAF/SIMPLE protein was identified as a putative binding partner for NS26 in a yeast two-hybrid screen. Here, we further characterized the association between NS26 and LITAF using in vivo and in vitro protein interaction assays. Soluble GST-NS26 and His6-LITAF were expressed and purified fromE. coli; recombinant NS26 tagged withmycand LITAF tagged with GFP were expressed inCtenopharyngon idelluskidney cells (CIK) by transient transfection experiments. A GST pulldown assay demonstrated that GST-tagged NS26 efficiently bound to His6-LITAF. Co-immunoprecipitation assays demonstrated that GCRV NS26 reciprocally precipitated endogenous LITAF in CIK cells. Double-immunofluorescent analyses revealedmyc-NS26 colocalized with GFP-LITAF in CIK cells. Taken together, the current in vitro and in vivo data demonstrated the interaction between cellular LITAF and GCRV NS26.