RNA-POLYMERASE OF INFLUENZA-VIRUS .11. PURIFICATION AND MOLECULAR-STRUCTURE OF RNA-POLYMERASE FROM INFLUENZA VIRUS-A/PR8
RNA-POLYMERASE OF INFLUENZA-VIRUS .11. PURIFICATION AND MOLECULAR-STRUCTURE OF RNA-POLYMERASE FROM INFLUENZA VIRUS-A/PR8
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DOI：
10.1093/oxfordjournals.jbchem.a123097
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发表时间：
1990-04-01
影响因子：
2.7
通讯作者：
ISHIHAMA, A
中科院分区：
文献类型：
--
作者：
HONDA, A;MUKAIGAWA, J;ISHIHAMA, A
用铯盐分级离心法从病毒颗粒中分离到依赖于RNA的流感病毒A/PR/8的RNA聚合酶。首先，用甘油梯度离心法分离病毒RNA-NP-P蛋白复合体，然后用氯化铯梯度离心法将NP从RNP中分离出来。用三氟乙酸铯(CsTFA)梯度离心法将P-RNA(P蛋白-病毒RNA)复合体进一步解离为P蛋白和病毒RNA。用甘油梯度离心法和免疫印迹法进一步分析P蛋白的性质。Pb1、Pb2和Pa三种P蛋白的沉淀速度与分子量约为25万Da的标记蛋白一样快。加入模板vRNA后，无RNA的P蛋白复合体表现出切割封顶的RNA和限制RNA合成的活性。当检测稳定表达三个P蛋白和NP蛋白的细胞系时，发现这三个P蛋白被针对单个P蛋白的抗体共沉淀。这些结果表明，流感病毒依赖RNA的RNA聚合酶是由三种P蛋白中的每一种组成的异源复合体，而无RNA的RNA聚合酶可以从病毒颗粒中以活性形式分离出来。此外，在没有vRNA的情况下，这三种P蛋白形成了一个复合体。
The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.