Barton D J, O'Donnell B J, Flanegan J B
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610-0245, USA.
EMBO J. 2001 Mar 15;20(6):1439-48. doi: 10.1093/emboj/20.6.1439.
A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.
脊髓灰质炎病毒RNA 5'末端的三叶草结构结合病毒和细胞蛋白。为了研究三叶草结构在脊髓灰质炎病毒复制中的作用，我们在HeLa S10翻译-复制反应中确定了三叶草突变如何影响脊髓灰质炎病毒RNA的稳定性、翻译和复制。在这些反应中，三叶草结构内的突变使病毒RNA不稳定。添加5' 7-甲基鸟苷帽完全恢复了突变RNA的稳定性，并且对其翻译没有影响。这些结果表明，5'三叶草结构通常保护未加帽的脊髓灰质炎病毒RNA不被细胞核酸酶快速降解。用加帽的突变RNA形成的起始前RNA复制复合物来测量负链合成。虽然突变RNA是稳定且有功能的mRNA，但它们不是负链RNA合成的活性模板。因此，5'三叶草结构是负链RNA合成起始所需的多功能顺式作用复制元件。我们提出了一个复制模型，其中病毒RNA的5'和3'末端相互作用形成一个环状核糖核蛋白复合物，该复合物调节脊髓灰质炎病毒RNA的稳定性、翻译和复制。
