In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
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DOI：
10.1371/journal.ppat.1000863
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发表时间：
2010-04-22
期刊：
影响因子：
6.7
通讯作者：
Decroly E
中科院分区：
文献类型：
--
作者：
Bouvet M;Debarnot C;Imbert I;Selisko B;Snijder EJ;Canard B;Decroly E
sars -冠状病毒（SARS-CoV）基因组的表达依赖于一组mrna的合成，这些mrna可能位于其5 '端，并指导受感染细胞中所有病毒蛋白的合成。16种病毒非结构蛋白（nsp1至nsp16）构成一个异常大的复制酶复合体，其中包括两种甲基转移酶，据推测参与病毒mRNA帽的形成。s -腺苷- l-蛋氨酸（AdoMet）依赖（鸟嘌呤- n7）-甲基转移酶（N7-MTase）活性最近归因于nsp14，而nsp16已被预测为AdoMet依赖（核苷-2 ' o）-甲基转移酶。在这里，我们在体外重建了完整的SARS-CoV mRNA帽甲基化。我们发现mRNA帽甲基化需要第三种病毒蛋白nsp10，它是完成RNA cap-1形成的必要触发因素。甲基化事件的专权序列由nsp14启动，它首先甲基化盖帽RNA转录物以产生cap- 70megpppa -RNA。后者被2'O-MTase nsp16及其激活剂nsp10复合物选择性地2 ' o -甲基化，产生cap-1 7megpppa2 ' ome - rna。此外，这两种活性的体外敏感抑制实验表明，在SARS-CoV感染细胞中具有活性的金羧酸对两种mtase的IC50值均在微摩尔范围内，为抗冠状病毒药物设计提供了有效的依据。2003年，一种新出现的冠状病毒（CoV）被确定为严重急性呼吸系统综合征（SARS）的病原。SARS-CoV利用含有多种病毒非结构蛋白的膜结合酶复合体复制和转录其大RNA基因组。RNA合成过程中的一个关键步骤是在新产生的病毒mrna上添加一个帽状结构，以确保它们被宿主细胞核糖体有效翻译。病毒通常要么从细胞mrna（例如流感病毒的“夺帽”）获得帽盖结构，要么利用它们自己的帽盖机制，就像冠状病毒的情况一样。病毒合成的mRNA帽在结构和功能上与细胞mRNA帽无法区分。在冠状病毒中，mRNA帽的甲基化似乎是必不可少的，因为病毒甲基转移酶nsp14或nsp16的突变使病毒无法存活。我们发现了SARS-CoV nsp10在mRNA帽甲基化过程中意想不到的关键作用，这是一种以前未知功能的蛋白质。Nsp10通过直接激活非活性的nsp16，诱导鸟嘌呤- n7甲基化盖顶rna的选择性2 ' o甲基化。这一发现允许在体外完全重构SARS-CoV mRNA帽甲基化序列，并为利用mRNA帽甲基转移酶作为抗冠状病毒药物设计的靶点开辟了道路。
SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5′ end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2′O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7MeGpppA-RNAs. The latter are then selectively 2′O-methylated by the 2′O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 7MeGpppA2′OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design. In 2003, an emerging coronavirus (CoV) was identified as the etiological agent of severe acute respiratory syndrome (SARS). SARS-CoV replicates and transcribes its large RNA genome using a membrane-bound enzyme complex containing a variety of viral nonstructural proteins. A critical step during RNA synthesis is the addition of a cap structure to the newly produced viral mRNAs, ensuring their efficient translation by host cell ribosomes. Viruses generally acquire their cap structure either from cellular mRNAs (e.g., “cap snatching” of influenza virus) or employ their own capping machinery, as is supposed to be the case for coronaviruses. mRNA caps synthesized by viruses are structurally and functionally undistinguishable from cellular mRNAs caps. In coronaviruses, methylation of mRNA caps seems to be essential, since mutations in viral methyltransferases nsp14 or nsp16 render non-viable virus. We have discovered an unexpected key role for SARS-CoV nsp10, a protein of previously unknown function, within mRNA cap methylation. Nsp10 induces selective 2′O-methylation of guanine-N7 methylated capped RNAs through direct activation of the otherwise inactive nsp16. This finding allows the full reconstitution of the SARS-CoV mRNA cap methylation sequence in vitro and opens the way to exploit the mRNA cap methyltransferases as targets for anti-coronavirus drug design.
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影响因子：
5
作者：
Gorbalenya AE;Enjuanes L;Ziebuhr J;Snijder EJ
通讯作者：
Snijder EJ
影响因子：
5
作者：
Imbert I;Snijder EJ;Dimitrova M;Guillemot JC;Lécine P;Canard B
通讯作者：
Canard B
影响因子：
5.4
作者：
BAKER, SC;YOKOMORI, K;LAI, MMC
通讯作者：
LAI, MMC
影响因子：
5.4
作者：
Donaldson, Eric F.;Sims, Amy C.;Baric, Ralph S.
通讯作者：
Baric, Ralph S.
影响因子：
5.4
作者：
Dong, Hongping;Ray, Debashish;Shi, Pei-Yong
通讯作者：
Shi, Pei-Yong