Clewley J P, Kennedy S I
J Gen Virol. 1976 Sep;32(3):395-411. doi: 10.1099/0022-1317-32-3-395.
A purification method for Semliki Forest virus-specified RNA-dependent RNA polymerase from BHK cells is described. The procedure entails (i) the preparation of a crude cell lysate by Dounce homogenization of cells 3-5 h post-infection, (ii) differential centrifugation to give a 15 000 g 'mitochondrial' pellet, (iii) equilibrium centrifugation on discontinuous sucrose gradients (Friedman et al. 1972) to give a membranous band of density 1-16 g/ml, (iv) solubilization with Triton N-101 and velocity centrifugation to give a 25S solubilized polymerase complex and (v) affinity chromatography through an oligo (dT)-cellulose matrix bearing immobilized 42S virus particle RNA. The overall purification was approx. 360-fold with a 5% recovery of activity. Of the various intermediate fractions in the purfication procedure, only the relatively crude post-nuclear supernatant fraction was competent to synthesize the major single-stranded RNAs found in infected cells. Other fractions incorporated precursor only into replicative intermediate (RI) or replicative from (RF). Analysis of the product RF showed that it was of the same size and could bind to the same extent to oligo (dT)-cellulose as the RF isolated directly from lysates of infected cells. Displacement hybridization and ribonuclease digestion suggested that the purified polymerase could only complete previously initiated progeny positive strands using negative strands as template and, even in its most highly purified form, was still tightly bound to its template. Analysis on polyacrylamide slab gels revealed the presence of three 35S-labelled polypeptides in the purified polymerase preparation, but a polypeptide which had identical electrophoretic mobility to the lowest mol. wt. polypeptide of the purified polymerase was also present in material from mock-fected cells which had been taken through the purification procedure. From these results we conclude that only two virus-specified polypeptides are present in the polymerase. A scheme for the synthesis of these polypeptides is presented in the accompanying paper.
本文描述了一种从BHK细胞中纯化塞姆利基森林病毒特异性RNA依赖性RNA聚合酶的方法。该步骤包括：（i）在感染后3 - 5小时通过Dounce匀浆法制备粗细胞裂解物；（ii）差速离心得到15000g的“线粒体”沉淀；（iii）在不连续蔗糖梯度上进行平衡离心（Friedman等人，1972年），得到密度为1.16g/ml的膜状带；（iv）用Triton N - 101溶解并进行速度离心，得到25S的溶解聚合酶复合物；（v）通过带有固定化42S病毒颗粒RNA的寡聚（dT）-纤维素基质进行亲和层析。总体纯化倍数约为360倍，活性回收率为5%。在纯化过程的各种中间组分中，只有相对粗制的核后上清组分能够合成感染细胞中发现的主要单链RNA。其他组分仅将前体掺入复制中间体（RI）或复制形式（RF）中。对产物RF的分析表明，其大小相同，并且与直接从感染细胞裂解物中分离的RF一样，能够在相同程度上结合到寡聚（dT）-纤维素上。置换杂交和核糖核酸酶消化表明，纯化的聚合酶只能以负链为模板完成先前起始的子代正链的合成，并且即使在其最高纯化形式下，仍与模板紧密结合。聚丙烯酰胺平板凝胶分析显示，纯化的聚合酶制剂中存在三种35S标记的多肽，但在经过纯化过程的 mock -感染细胞的材料中也存在一种与纯化聚合酶最低分子量多肽具有相同电泳迁移率的多肽。根据这些结果，我们得出结论，聚合酶中仅存在两种病毒特异性多肽。这些多肽的合成方案见随附论文。
