A conserved motif in region V of the large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential for mRNA capping
A conserved motif in region V of the large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential for mRNA capping
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DOI：
10.1128/jvi.02107-07
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发表时间：
2008-01-01
影响因子：
5.4
通讯作者：
Whelan, Sean P. J.
中科院分区：
文献类型：
--
作者：
Lij, Jianrong;Rahmeh, Amal;Whelan, Sean P. J.
非节段负义（NNS） RNA病毒通过一种非常规机制盖住其mRNA。具体来说，5 '单磷酸mRNA通过大聚合酶蛋白L和mRNA之间的共价中间体反应转移到GTP衍生的GDP中。这种多核糖核苷转移酶活性与所有其他由RNA三磷酸酶和鸟苷转移酶催化的盖帽反应形成对比。在这些反应中，5 '二磷酸mRNA通过共价酶-GMP中间体被GMP转移所限制。RNA胍基转移酶通常具有一个KxDG基序，其中赖氨酸形成这种共价中间体。与NNS RNA病毒独特的旋盖机制一致，L中不存在这样的基序。为了确定旋盖所需的L蛋白残基，我们用高度纯化的成分重建了NNS RNA原型病毒水泡性口炎病毒的旋盖反应。利用一组在NNS RNA病毒L蛋白中普遍保守的残基上进行单氨基酸替换的L蛋白，我们定义了一个新的基序GxxT[n]HR，它存在于L蛋白的保守区V中，对于这种非常规的mRNA帽形成机制至关重要。
Nonsegmented negative-sense (NNS) RNA viruses cap their mRNA by an unconventional mechanism. Specifically, 5 ' monophosphate mRNA is transferred to GDP derived from GTP through a reaction that involves a covalent intermediate between the large polymerase protein L and mRNA. This polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an RNA triphosphatase and guanylyltransferase. In these reactions, a 5 ' diphosphate mRNA is capped by transfer of GMP via a covalent enzyme-GMP intermediate. RNA guanylyltransferases typically have a KxDG motif in which the lysine forms this covalent intermediate. Consistent with the distinct mechanism of capping employed by NNS RNA viruses, such a motif is absent from L. To determine the residues of L protein required for capping, we reconstituted the capping reaction of the prototype NNS RNA virus, vesicular stomatitis virus, from highly purified components. Using a panel of L proteins with single-amino-acid substitutions to residues universally conserved among NNS RNA virus L proteins, we define a new motif, GxxT[n]HR, present within conserved region V of L protein that is essential for this unconventional mechanism of mRNA cap formation.