Osman T A, Buck K W
Department of Biology, Imperial College of Science, Technology and Medicine, London, United Kingdom.
J Virol. 1997 Aug;71(8):6075-82. doi: 10.1128/JVI.71.8.6075-6082.1997.
A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.
一种经蔗糖密度梯度纯化的、含有内源性RNA模板的膜结合烟草花叶病毒（番茄株系L）（TMV-L）RNA聚合酶，用牛磺脱氧胆酸钠可有效地使其溶解。溶解导致正链6.4-kb基因组长度单链RNA（ssRNA）的合成增加，而6.4-kbp双链RNA（dsRNA）的产生减少至接近检测极限的水平。溶解的TMV-L RNA聚合酶通过DEAE-生物凝胶柱和High Q柱色谱法进行纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和银染分析表明，纯化的RNA聚合酶制剂始终含有分子量为183、126、56、54和50 kDa的蛋白质，这些蛋白质在健康植物的等量材料中未发现。蛋白质印迹法表明，其中两个最大的蛋白质是TMV-L编码的183-kDa和126-kDa复制蛋白，56-kDa蛋白质与酵母eIF-3的RNA结合亚基54.6-kDa GCD10蛋白相关。126-kDa、183-kDa和56-kDa蛋白质通过抗TMV-L 126-kDa蛋白的抗体和抗GCD10蛋白的抗体进行共免疫亲和选择。抗体连接的聚合酶测定表明，活性TMV-L RNA聚合酶与抗TMV-L 126-kDa蛋白的抗体和抗GCD10蛋白的抗体结合。针对GCD10蛋白的抗体抑制了模板依赖性膜结合RNA聚合酶对基因组长度ssRNA和dsRNA的合成，并且通过预先添加GCD10蛋白可逆转这种抑制作用。
