RNA-POLYMERASE OF INFLUENZA-VIRUS .3. ISOLATION OF RNA-POLYMERASE RNA COMPLEXES FROM INFLUENZA-VIRUS PR8
RNA-POLYMERASE OF INFLUENZA-VIRUS .3. ISOLATION OF RNA-POLYMERASE RNA COMPLEXES FROM INFLUENZA-VIRUS PR8
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DOI：
10.1093/oxfordjournals.jbchem.a134254
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发表时间：
1983-01-01
影响因子：
2.7
通讯作者：
ISHIHAMA, A
中科院分区：
文献类型：
--
作者：
KAWAKAMI, K;ISHIHAMA, A
采用不连续的硫酸铯、甘油和NP-40三重梯度分离法，从经过洗涤剂处理的流感病毒PR8中分离出具有rna合成活性的核糖核蛋白（RNP）核心，分为无M蛋白和M蛋白相关两部分。通过磷纤维素柱层析将无m的RNP分离为P蛋白含量不同的A、B两种主要RNP形式，而与m相关的RNP只产生低P含量的B型RNP。从高p含量的Form-A RNP开始，通过硫酸铯平衡离心分离得到一个几乎不含NP蛋白的RNA-P蛋白复合物。该复合物仅含有3个P蛋白（P1， P2和P3），在体外不添加外源模板的情况下仍具有催化RNA合成的活性，表明NP蛋白不需要催化RNA合成。分离的RNA- p蛋白复合物的RNA合成依赖于ApG或带帽RNA引物，需要4个三磷酸核糖核苷作为底物。该反应的RNA产物可与病毒RNA杂交。在RNase处理或氯化铯平衡离心后，用凝胶甘油梯度离心从RNA中分离出各一个P蛋白复合物。
Ribonucleoprotein (RNP) cores with RNA-synthesizing activity were prepared in 2 fractions, M protein-free and M protein-associated, from detergent-treated influenza virus PR8 by centrifugation through a discontinuous triple gradient of cesium sulfate, glycerol and NP-40. The M-free RNP was fractionated by phosphocellulose column chromatography into 2 major RNP forms, A and B, which differed in the content of P proteins, while the M-associated RNP gave only the low P-content Form-B RNP. Starting from the high P-content Form-A RNP, an RNA-P protein complex virtually free from NP protein was isolated by cesium sulfate equilibrium centrifugation. The complex, containing only 3 P proteins (P1, P2 and P3), was still active in catalyzing RNA synthesis in vitro without addition of exogenous template, indicating that NP protein is not required for the catalysis of RNA synthesis. RNA synthesis by the isolated RNA-P protein complex was dependent on either ApG or capped RNA primers, and required 4 ribonucleoside triphosphates as substrates. The RNA product in this reaction was hybridizable to viral RNA. A complex of one each of the 3 P proteins was separated from RNA by gel glycerol gradient centrifugation after RNase treatment or cesium chloride equilibrium centrifugation.