Differential roles of viral RNA and cRNA in functional modulation of the influenza virus RNA polymerase
Differential roles of viral RNA and cRNA in functional modulation of the influenza virus RNA polymerase
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DOI：
10.1074/jbc.m102856200
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发表时间：
2001-08-17
影响因子：
4.8
通讯作者：
Ishihama, A
中科院分区：
文献类型：
--
作者：
Honda, A;Endo, A;Ishihama, A
流感病毒的RNA依赖性RNA聚合酶由三种病毒P蛋白（PB1、PB2和PA）组成，并参与RNA基因组的转录和复制。对于这种多功能酶的分子解剖，我们已经建立了一个同时表达的三个P蛋白在培养的昆虫细胞中使用重组杆状病毒。为了纯化P蛋白复合物，PA蛋白表达为在其N末端添加有组氨酸标签的融合物。通过亲和层析，从病毒感染细胞的核提取物中分离出由三种P蛋白组成的复合物。亲和纯化的3P复合物显示了加帽RNA结合、加帽RNA切割、病毒模型RNA结合、模型RNA指导的RNA合成和新合成RNA的多聚腺苷酸化的活性。我们的结论是，在重组杆状病毒感染的昆虫细胞中形成了具有转录酶催化特异性的病毒RNA聚合酶的功能形式。使用无病毒RNA的3P复合物，我们发现加帽RNA切割在vRNA而不是cRNA存在下发生，表明vRNA作为病毒RNA聚合酶特异性控制的调节因子以及转录模板发挥作用。使用模型RNA模板的变体形式分析指导RNA聚合酶功能表达的RNA的结构元件。
The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multifunctional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA binding, capped RNA cleavage, viral model RNA binding, model RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirus-infected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vRNA but not of cRNA, indicating that the vRNA functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates.