Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic.
PLoS Pathog. 2020 Dec 2;16(12):e1009100. doi: 10.1371/journal.ppat.1009100. eCollection 2020 Dec.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.
严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是 2019 年冠状病毒病 (COVID-19) 的病原体。SARS-CoV-2 是一种单链正链 RNA 病毒。与其他冠状病毒一样，SARS-CoV-2 的基因组非常大，编码四个结构蛋白和十六个非结构蛋白。结构核衣壳磷蛋白 N 对于将病毒基因组与病毒膜连接至关重要。N 端 RNA 结合 (N-NTD) 和 C 端二聚化结构域都参与捕获 RNA 基因组，并且这些结构域之间的无规卷曲区域将核糖核蛋白复合物锚定在病毒膜上。在这里，我们使用 NMR 光谱法表征了 N-NTD 的结构及其与 RNA 的相互作用。我们观察到 N-NTD 表面上存在一个带正电荷的峡谷，它可能像其他冠状病毒一样充当潜在的 RNA 结合位点。随后使用单链和双链 RNA 的 NMR 滴定实验揭示了一个更加广泛的 U 形 RNA 结合裂隙，其中排列着规则分布的精氨酸和赖氨酸。突变分析支持的 NMR 数据使我们能够构建 N-NTD/RNA 复合物的杂交原子模型，从而深入了解 RNA 识别。
