ROLE OF 2 OF THE INFLUENZA-VIRUS CORE P-PROTEINS IN RECOGNIZING CAP-1 STRUCTURES (M7GPPPNM) ON RNAS AND IN INITIATING VIRAL-RNA TRANSCRIPTION
ROLE OF 2 OF THE INFLUENZA-VIRUS CORE P-PROTEINS IN RECOGNIZING CAP-1 STRUCTURES (M7GPPPNM) ON RNAS AND IN INITIATING VIRAL-RNA TRANSCRIPTION
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DOI：
10.1073/pnas.78.12.7355
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发表时间：
1981-01-01
期刊：
影响因子：
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通讯作者：
KRUG, RM
中科院分区：
文献类型：
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作者：
ULMANEN, I;BRONI, BA;KRUG, RM
纯化的流感病毒核心催化病毒RNA转录的整个过程，包括对含有cap 1 （m7GpppNm）结构的异源RNA进行核内裂解裂解，生成10-13个核苷酸长的带帽引物，通过将鸟苷残基结合到引物上开始转录，以及病毒mRNA的延伸。为了确定哪些病毒核心蛋白（核衣壳蛋白，P1， P2或P3）识别RNA引物上的cap 1结构，在没有三磷酸核糖核苷的情况下，病毒核心使用标记为cap 1结构的32P引物RNA进行内切酶反应，并进行紫外线照射诱导交联。在1维和2维凝胶电泳中，标记的帽与一个蛋白交联，该蛋白具有与P3蛋白相似的流动性，P3蛋白是2个碱性P蛋白中较小的一个，这表明该交联蛋白是P3。与含有或缺乏cap 1结构的未标记RNA的竞争实验证实，该蛋白识别RNA上的cap 1结构。在整个转录反应过程中，即使在病毒mRNA分子被拉长之后，这种蛋白仍然与帽相关。为了鉴定通过鸟苷残基结合到引物片段上催化转录起始的病毒核心蛋白，转录反应在[.alpha]存在的情况下由病毒核心进行。-32P]GTP作为唯一的三磷酸核糖核苷，带未标记的引物RNA，并进行紫外线照射。在一维和二维凝胶电泳中，标记的鸟苷残基与具有类似于P1（2个碱性P蛋白中较大的一个）的迁移率的蛋白质交联。将该蛋白与标记的鸟苷残基紧密结合从而发生交联所需的转录反应条件表明，这种结合很可能是在鸟苷残基被结合到引物上时发生的。病毒P1蛋白显然催化这种结合，从而启动转录。
Purified influenza viral cores catalyze the entire process of viral RNA trascription, which includes the endonucleolytic cleavage of heterologous RNA containing cap 1 (m7GpppNm) structures to generate capped primers 10-13 nucleotides long, the initiation of transcription via the incorporation of a guanosine residue onto the primers and elongation of the viral mRNA. To identify which viral core protein (nucleocapsid protein, P1, P2 or P3) recognizes the cap 1 structure on the RNA primer, endonuclease reactions were carried out by viral cores in the absence of ribonucleoside triphosphates using a primer RNA labeled in its cap 1 structure with 32P, and the preparation was UV-irradiated to induce crosslinking. The labeled cap was crosslinked to a protein that had a mobility similar to that of the P3 protein, the smaller of the 2 basic P proteins, in both 1- and 2-dimensional gel electrophoresis, suggesting that this crosslinked protein is P3. Competition experiments with unlabeled RNA containing or lacking a cap 1 structure established that this protein recognizes the cap 1 straucture on RNA. This protein remained associated with the cap throughout the transcription reaction, even after the viral mRNA molecules were elongated. To identify the viral core protein that catalyzes the initiation of transcription via the incorporation of a guanosine residue onto primer fragments, transcription reactions were carried out by viral cores in the presence of [.alpha.-32P]GTP as the only ribonucleoside triphosphate and with an unlabeled primer RNA and the preparation was UV-irradiated. A labeled guanosine residue was crosslinked to a protein that had a mobility similar to that of P1, the larger of the 2 basic P proteins, in both 1- and 2-dimensional gel electrophoresis. The transcription reaction conditions required to bring this protein in close association with a labeled guanosine residue so that crosslinking could occur indicated that this association most likely occurred coincident with the guanosine residue''s being incorporated onto the primer. The viral P1 protein apparently catalyzes this incorporation and hence initiates transcription.