Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase
Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase
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功能筛选显示 SARS 冠状病毒非结构蛋白 nsp14 是一种新型帽 N7 甲基转移酶
DOI：
10.1073/pnas.0808790106
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发表时间：
2009-03-03
影响因子：
11.1
通讯作者：
Guo, Deyin
中科院分区：
文献类型：
--
作者：
Chen, Yu;Cai, Hui;Guo, Deyin
N7-甲基鸟苷（m7 G）帽是真核生物mRNA的定义性结构特征。大多数在细胞质中复制的真核病毒，包括冠状病毒，都进化出了给它们的RNA加帽的策略。在这份报告中，我们使用酵母遗传系统的功能筛选的帽形成酶编码的严重急性呼吸综合征（SARS）冠状病毒，并确定了非结构蛋白（nsp）14作为（鸟嘌呤-N7）-甲基转移酶（N7-MTase）在体内酵母细胞和体外使用纯化的酶和RNA底物。有趣的是，冠状病毒nsp 14先前被表征为3′至5′核糖核酸外切酶，通过突变分析，我们将N7-MTase结构域定位到nsp 14的羧基末端部分，该部分在基于结构的序列比对中显示出与细胞N7-MTase保守的特征。核糖核酸外切酶活性位点是缺失的，但核糖核酸外切酶结构域是N7-MTase活性所必需的。冠状病毒nsp 14中两个功能结构域的这种组合表明，它可能代表一种新形式的RNA加工酶。在复制子系统中的突变分析表明，N7-MTase活性对于SARS病毒复制/转录是重要的，因此可以用作有吸引力的药物靶标来开发用于控制冠状病毒（包括致命的SARS病毒）的抗病毒药物。此外，RNA生命中的N7-MTase可以代替DNA生命中的N7-MTase发挥作用的观察结果为抗病毒药物筛选提供了有趣的进化见解和实际可能性。
The N7-methylguanosine (m7G) cap is the defining structural feature of eukaryotic mRNAs. Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. In this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes encoded by severe acute respiratory syndrome (SARS) coronavirus and identified the nonstructural protein (nsp) 14 of SARS coronavirus as a (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast cells and in vitro using purified enzymes and RNA substrates. Interestingly, coronavirus nsp14 was previously characterized as a 3′-to-5′ exoribonuclease, and by mutational analysis, we mapped the N7-MTase domain to the carboxy-terminal part of nsp14 that shows features conserved with cellular N7-MTase in structure-based sequence alignment. The exoribonuclease active site was dispensable but the exoribonuclease domain was required for N7-MTase activity. Such combination of the 2 functional domains in coronavirus nsp14 suggests that it may represent a novel form of RNA-processing enzymes. Mutational analysis in a replicon system showed that the N7-MTase activity was important for SARS virus replication/transcription and can thus be used as an attractive drug target to develop antivirals for control of coronaviruses including the deadly SARS virus. Furthermore, the observation that the N7-MTase of RNA life could function in lieu of that in DNA life provides interesting evolutionary insight and practical possibilities in antiviral drug screening.