Rottier P J, Spaan W J, Horzinek M C, van der Zeijst B A
J Virol. 1981 Apr;38(1):20-6. doi: 10.1128/JVI.38.1.20-26.1981.
We have purified the seven virus-specific RNAs which were previously shown to be induced in Sac(-) cells upon infection with mouse hepatitis virus strain A59 (W. J. M. Spaan, P. J. M. Rottier, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 108:424-434, 1981). The individual RNAs, prepared by agarose gel electrophoresis of the polyadenylated RNA fraction from infected cells, were obtained pure, except for the preparations of RNAs 4, 5, and 6, which contained some contamination of RNA 7. The RNAs were microinjected into Xenopus laevis oocytes, and after incubation of these cells in the presence of [35S]methionine, the proteins synthesized were analyzed by polyacrylamide gel electrophoresis. Whereas no translation products of RNAs 1, 2, 4, and 5 were detected, the synthesis of virus-specific polypeptides coded by RNAs 3, 6, and 7 was observed. RNA 7 (0.6 X 10(6) daltons) directed the synthesis of a 54,000-molecular-weight polypeptide which comigrated with viral nucleocapsid protein and which was immunoprecipitated by antiserum from mice that had been infected with the virus. RNA 6 (0.9 X 10(6) daltons) directed the synthesis of three polypeptides with molecular weights of 24,000, 25,500, and 26,500, which migrated with the same electrophoretic mobilities as three low-molecular-weight virion polypeptides. After injection of RNA 3 (3.0 X 10(6) daltons), a polypeptide with a molecular weight of about 150,000 was immunoprecipitated. This polypeptide had no counterpart in the virion, but comigrated with a virus-specific glycoprotein present in infected cells which is immunoprecipitated by a rabbit antiserum against the mouse hepatitis virus strain A59 structural proteins. This antiserum could also immunoprecipitate the translation products of RNAs 3, 6, and 7. These results indicate that RNAs 3, 6, and 7 encode viral structural proteins. The significance of the data with respect to the strategy of coronavirus replication is discussed.
我们已经纯化了七种病毒特异性RNA，之前的研究表明，在用A59小鼠肝炎病毒感染无病毒（Sac(-)）细胞后，这些RNA会被诱导产生（W. J. M. Spaan、P. J. M. Rottier、M. C. Horzinek和B. A. M. van der Zeijst，《病毒学》108:424 - 434，1981）。通过对感染细胞的聚腺苷酸化RNA组分进行琼脂糖凝胶电泳制备的各个RNA，除了RNA 4、5和6的制备物中含有一些RNA 7的污染外，均获得了纯品。将这些RNA显微注射到非洲爪蟾卵母细胞中，在[35S]甲硫氨酸存在的情况下孵育这些细胞后，通过聚丙烯酰胺凝胶电泳分析合成的蛋白质。虽然未检测到RNA 1、2、4和5的翻译产物，但观察到了由RNA 3、6和7编码的病毒特异性多肽的合成。RNA 7（0.6×10^6道尔顿）指导合成了一种分子量为54,000的多肽，该多肽与病毒核衣壳蛋白迁移率相同，并且能被感染该病毒的小鼠血清免疫沉淀。RNA 6（0.9×10^6道尔顿）指导合成了三种分子量分别为24,000、25,500和26,500的多肽，它们与三种低分子量病毒粒子多肽具有相同的电泳迁移率。注射RNA 3（3.0×10^6道尔顿）后，一种分子量约为150,000的多肽被免疫沉淀。这种多肽在病毒粒子中没有对应物，但与感染细胞中存在的一种病毒特异性糖蛋白迁移率相同，该糖蛋白能被抗A59小鼠肝炎病毒结构蛋白的兔血清免疫沉淀。这种血清还能免疫沉淀RNA 3、6和7的翻译产物。这些结果表明，RNA 3、6和7编码病毒结构蛋白。文中讨论了这些数据对于冠状病毒复制策略的意义。
