Laboratory of Structural Biology, School of Medicine, Tsinghua University, Beijing, China.
State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
J Virol. 2018 Oct 29;92(22). doi: 10.1128/JVI.00893-18. Print 2018 Nov 15.
Nonstructural protein 15 (Nsp15) encoded by coronavirus (CoV) is a nidoviral uridylate-specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural information on this crucial protein from the Middle East respiratory syndrome CoV (MERS-CoV), which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we determined the crystal structure of MERS-CoV Nsp15 at a 2.7-Å resolution and performed the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with that of other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A, and N157A, decrease thermostability, decrease affinity of binding with RNA, and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 was found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15 Taking the findings together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency, or the possible association with other nonstructural proteins. The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and the Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.
冠状病毒（CoV）编码的非结构蛋白 15（Nsp15）是一种病毒内切酶（NendoU），在病毒的生命周期中起着至关重要的作用。中东呼吸综合征冠状病毒（MERS-CoV）的这种关键蛋白的结构信息缺乏，该病毒具有致命性，已在全球范围内引起严重的呼吸道疾病。在这里，我们确定了 MERS-CoV Nsp15 的晶体结构，分辨率为 2.7Å，并进行了相关的生化测定以研究 NendoU 活性如何受到调节。尽管整体结构是保守的，但与同源物相比，MERS-CoV Nsp15 具有独特而新颖的特征。残基 F285 的丝氨酸取代，与其他同源物相比，该残基具有芳香侧链，会干扰 RNA 结合，从而提高了催化活性。位于寡聚化界面上的改变六聚化的残基的突变，即 N38A、Y58A 和 N157A，降低了热稳定性，降低了与 RNA 的结合亲和力，并降低了 Nsp15 的 NendoU 活性。相比之下，突变体 D39A 表现出更高的活性和更高的底物结合能力。重要的是，发现 Nsp8 与单体和六聚体 Nsp15 相互作用。Nsp7/Nsp8 复合物对 Nsp15 具有更高的结合亲和力。此外，Nsp8 和 Nsp7/Nsp8 复合物还增强了六聚体 Nsp15 的 NendoU 活性。综上所述，这项工作首次提供了证据，证明 Nsp15 的活性如何通过催化残基、寡聚体组装、RNA 结合效率或与其他非结构蛋白的可能关联来进行功能调节。致死性中东呼吸综合征冠状病毒（MERS-CoV）和严重急性呼吸综合征冠状病毒（SARS-CoV）对人类构成严重威胁。冠状病毒编码的内切酶 Nsp15 在病毒感染和发病机制中起重要作用。本研究确定了 MERS-CoV Nsp15 的结构，并展示了该蛋白的催化活性如何受到潜在调节，从而为开发抗病毒药物提供了结构和功能证据。我们还假设引物酶样蛋白 Nsp8 和 Nsp7/Nsp8 复合物可能与 Nsp15 相互作用并影响酶活性。这有助于理解 Nsp15 与病毒复制和转录机制的关联。
