Subcellular localisation, protein interactions, and RNA binding of potato mop-top virus triple gene block proteins
Subcellular localisation, protein interactions, and RNA binding of potato mop-top virus triple gene block proteins
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DOI：
10.1006/viro.2002.1435
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发表时间：
2002-06-20
期刊：
影响因子：
3.7
通讯作者：
Torrance, L
中科院分区：
文献类型：
--
作者：
Cowan, GH;Lioliopoulou, F;Torrance, L
研究了马铃薯拖把顶病毒（PMTV）三重基因阻断蛋白（TGBp）的亚细胞定位、蛋白质相互作用和RNA结合。利用重组烟草花叶病毒（TMV）载体分别表达了N端融合有或没有绿色荧光蛋白的13-kDa（TGBp 2）和21-kDa（TGBp 3）蛋白以及51-kDa蛋白（TGBp 1）。荧光图像和Western免疫印迹实验的重组TMV感染的本氏烟草细胞表明，TGBp 2和TGBp 3与细胞内膜和TGBp 3与细胞壁，可能位于靠近胞间连丝，在Western印迹中，TGBp 1检测到含有细胞壁的组分和那些丰富的细胞器和膜结构。在酵母双杂交实验中证明了与所有三种蛋白质的自相互作用，并且在TGBp 2和TGBp 3之间发现了异源相互作用。在不同的TGBp之间没有发现另外的异源相互作用，并且在体外结合测定中没有检测到。TGBp 1和TGBp 2而不是TGBp 3显示以序列非特异性方式结合ssRNA。结果支持TGBp 2和TGBp 3促进核糖核蛋白复合物向胞间连丝的递送和定位的模型。然而，该过程是由RNA-蛋白质而不是与病毒RNA复合的TGBp 1和膜定位的TGBp 2之间的蛋白质-蛋白质相互作用促进的，（C）2002 Elsevier Science（USA）。
Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied, The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-Infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata, In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata, However, the process is facilitated by RNA-protein rather than protein protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2, (C) 2002 Elsevier Science (USA).