Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.
Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.
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DOI：
10.1371/journal.ppat.1002294
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发表时间：
2011-10
期刊：
影响因子：
6.7
通讯作者：
Guo D
中科院分区：
文献类型：
--
作者：
Chen Y;Su C;Ke M;Jin X;Xu L;Zhang Z;Wu A;Sun Y;Yang Z;Tien P;Ahola T;Liang Y;Liu X;Guo D
5′-帽结构是真核生物mRNA的一个显著特征，真核病毒通常会修饰病毒RNA的5′-端以模拟细胞mRNA结构，这对RNA稳定性、蛋白质翻译和病毒免疫逃逸至关重要。SARS冠状病毒（SARS-CoV）编码两个依赖S-腺苷-L-甲硫氨酸（SAM）的甲基转移酶（MTase），分别在nsp 14 N7-MTase和nsp 16 2′-O-MTase的催化下，将RNA帽的鸟苷-N7和核糖2′-O位甲基化。SARS-CoV的一个独特特征是nsp 16需要非结构蛋白nsp 10作为刺激因子来执行其MTase活性。本文报道了SARS-CoV 2′-O-MTase的生物化学特性和与甲基供体SAM结合的nsp 16/nsp 10复合物的晶体结构。我们发现SARS-CoV nsp 16 MTase甲基化m7 GpppA-RNA，但不甲基化m7 GpppG-RNA，这与nsp 14 MTase以不依赖于序列的方式起作用相反。我们证明了nsp 16结合m7 GpppA-RNA底物和SAM辅因子需要nsp 10。结构分析表明，nsp 16具有MTase的典型骨架结构，与nsp 10以1∶1的比例结合。nsp 16/nsp 10相互作用界面的结构表明，nsp 10可以稳定SAM结合口袋和扩展nsp 16的底物RNA结合沟，与生化测定中的发现一致。这些结果表明，nsp 16/nsp 10界面可能代表比病毒MTase活性位点更好的药物靶点，用于开发高度特异性的抗冠状病毒药物。真核生物mRNA的显著特征是存在mRNA稳定性和蛋白质翻译所需的甲基化帽结构。由于所有病毒都使用细胞核糖体进行蛋白质翻译，因此大多数细胞质复制的真核病毒（包括冠状病毒）都进化出了加帽其RNA的策略。最近的研究表明，病毒RNA帽结构中的核糖2′-O-甲基化在病毒逃避先天免疫识别中起着重要作用。SARS冠状病毒编码的2′-O-甲基转移酶（2′-O-MTase）由催化亚基nsp 16和刺激亚基nsp 10两个亚基组成，不同于其他已知的2′-O-MTase，它不依赖于配偶体。在这里，我们表明，nsp 10的作用是促进nsp 16结合加帽RNA底物和甲基供体S-腺苷-L-甲硫氨酸（SAM）。我们解析了nsp 16/nsp 10/SAM复合物的晶体结构，结构分析揭示了分子间相互作用的细节，并表明nsp 10可以稳定SAM结合口袋并扩展加帽的RNA结合沟。nsp 16/nsp 10的相互作用界面对于冠状病毒是独特的，因此可能为开发用于控制冠状病毒（包括致命的SARS冠状病毒）的特异性抗病毒药物提供有吸引力的靶点。
The 5′-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5′-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2′-O positions, catalyzed by nsp14 N7-MTase and nsp16 2′-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2′-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs. The distinctive feature of eukaryotic mRNAs is the presence of methylated cap structure that is required for mRNA stability and protein translation. As all viruses employ cellular ribosomes for protein translation, most cytoplasmically replicating eukaryotic viruses including coronaviruses have evolved strategies to cap their RNAs. It was shown very recently that ribose 2′-O-methylation in the cap structure of viral RNAs plays an important role in viral escape from innate immune recognition. The 2′-O-methyltransferase (2′-O-MTase) encoded by SARS coronavirus is composed of two subunits, the catalytic subunit nsp16 and the stimulatory subunit nsp10, which is different from all other known 2′-O-MTases that are partner-independent. Here we show that the role of nsp10 is to promote nsp16 to bind capped RNA substrate and the methyl donor S-adenosyl-L-methionine (SAM). We solved the crystal structure of the nsp16/nsp10/SAM complex, and the structural analysis revealed that the details of the inter-molecular interactions and indicated that nsp10 may stabilize the SAM-binding pocket and extend the capped RNA-binding groove. The interaction interface of nsp16/nsp10 is unique for coronaviruses and thus may provide an attractive target for developing specific antiviral drugs for control of coronaviruses including the deadly SARS coronavirus.
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影响因子：
6.7
作者：
Decroly E;Debarnot C;Ferron F;Bouvet M;Coutard B;Imbert I;Gluais L;Papageorgiou N;Sharff A;Bricogne G;Ortiz-Lombardia M;Lescar J;Canard B
通讯作者：
Canard B
DOI：
10.1107/s0907444907046148
发表时间：
2007-11
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Afonine PV;Grosse-Kunstleve RW;Adams PD;Lunin VY;Urzhumtsev A
通讯作者：
Urzhumtsev A
影响因子：
4.8
作者：
Hager, J;Staker, BL;Jakob, U
通讯作者：
Jakob, U
影响因子：
5.6
作者：
Bollati, Michela;Milani, Mario;Bolognesi, Martino
通讯作者：
Bolognesi, Martino
病毒mRNA帽的2'-O甲基化通过IFIT家族成员逃避了宿主的限制。
影响因子：
64.8
作者：
通讯作者：
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