Template Recognition Mechanisms by Replicase Proteins Differ between Bipartite Positive-Strand Genomic RNAs of a Plant Virus
Template Recognition Mechanisms by Replicase Proteins Differ between Bipartite Positive-Strand Genomic RNAs of a Plant Virus
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DOI：
10.1128/jvi.01754-10
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发表时间：
2011-01-01
影响因子：
5.4
通讯作者：
Okuno, Tetsuro
中科院分区：
文献类型：
--
作者：
Iwakawa, Hiro-oki;Mine, Akira;Okuno, Tetsuro
病毒复制酶蛋白对RNA模板的识别是所有RNA病毒复制过程中的关键步骤之一。然而，这种现象背后的机制，包括被病毒复制酶蛋白识别的初级RNA元素，还没有得到很好的理解。在这里，我们在无细胞病毒翻译/复制系统中使用膜分离和蛋白质- rna共免疫沉淀的适体下拉实验来研究病毒复制酶蛋白如何识别红三叶草坏死花叶病毒（RCNMV）的双部基因组rna。RCNMV复制酶蛋白与位于RNA2 3'非翻译区（UTR）的y形RNA元件（YRE）特异性结合，该元件也与含有病毒和宿主蛋白的480 kda复制酶复合物相互作用。复制酶- yre相互作用将RNA2招募到膜部分。相反，RNA1片段不能与反式中提供的复制酶蛋白相互作用。蛋白- rna共免疫沉淀试验的结果表明，RNA1与复制酶蛋白相互作用，并结合它们的翻译。因此，复制酶采用的初始模板识别机制在RCNMV双基因组RNA之间存在差异，RNA元件是差异复制机制的主要决定因素。
Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3' untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.