Binding of the Methyl Donor S-Adenosyl-L-Methionine to Middle East Respiratory Syndrome Coronavirus 2′-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10
Binding of the Methyl Donor S-Adenosyl-L-Methionine to Middle East Respiratory Syndrome Coronavirus 2′-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10
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DOI：
10.1128/jvi.02217-16
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发表时间：
2017-03-01
影响因子：
5.4
通讯作者：
Decroly, Etienne
中科院分区：
文献类型：
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作者：
Aouadi, Wahiba;Blanjoie, Alexandre;Decroly, Etienne
中东呼吸综合征冠状病毒（MERS-CoV）非结构蛋白16 （nsp16）是一种s -腺苷- l-蛋氨酸（SAM）依赖的2'- o -甲基转移酶（2'-O-MTase），被认为是使病毒RNA帽结构的第一个转录核苷酸（N-1）的核糖2'-OH甲基化。2'-O-MTase活性受nsp10调控。2'-O甲基化通过细胞先天免疫机制和干扰素刺激的IFIT-1蛋白的病毒翻译抑制来阻止病毒检测。为了揭示nsp10/nsp16 2'-O-MTase活性的调控，我们使用纯化的MERS-CoV nsp16和nsp10。首先，我们发现nsp16招募了n7甲基化的帽RNA和SAM。SAM结合促进酶活性nsp10/nsp16复合物的组装，该复合物通过2'-OH甲基化N1以SAM依赖的方式将（7m）GpppG（cap-0）转化为（7m）GpppG(2' om) (cap-1) RNA。随后释放的SAH加速了nsp10/nsp16的解离，从而刺激了反应的转换。通过丙氨酸诱变和RNA结合实验，鉴定了参与RNA识别形成RNA结合槽的nsp16残基（K46、K170、E203、D133、R38、Y47和Y181）和cap-0结合位点（Y30、Y132和H174）。最后，我们发现nsp10/nsp16 2'-O-MTase活性对已知的MTase抑制剂（如sininefungin和cap类似物）敏感。MERS-CoV 2'-O- mtase的这一特性是开发抑制cap 2'-O甲基化和恢复宿主抗病毒反应的分子的初步步骤。MERS-CoV编码cap 2'- o -甲基转移酶，将cap-0转化为cap-1结构，以阻止细胞先天免疫机制检测病毒。我们报道了MERS-CoV 2'- o -甲基转移酶的生化特性，nsp10作为nsp16 2'- o -甲基转移酶的变抗激活剂可能通过增强RNA结合亲和力来刺激该酶。此外，我们发现SAM促进了活性nsp10/nsp16复合物的形成。相反，帽甲基化后，在低细胞内SAH浓度下，cap-1 RNA释放和nsp10/nsp16复合物解离加速了反应的周转。这些结果表明，SAM/SAH平衡是2'- o -甲基转移酶活性的调节因子，并提出了SAH水解酶抑制剂可能干扰冠状病毒复制周期的可能性。在这项工作中开发的酶和RNA结合试验也用于鉴定参与cap-0 RNA识别的nsp16残基，并了解已知甲基转移酶抑制剂的作用模式。
The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-L-methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase) that is thought to methylate the ribose 2'-OH of the first transcribed nucleotide (N-1) of viral RNA cap structures. This 2'-O-MTase activity is regulated by nsp10. The 2'-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2'-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted (7m)GpppG (cap-0) into (7m)GpppG(2'Om) (cap-1) RNA by 2'-OH methylation of N1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2'-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2'-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2'-O methylation and to restore the host antiviral response.IMPORTANCE MERS-CoV codes for a cap 2'-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2'O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2'-O-methyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2'-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.