Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455.
Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455.
Proc Natl Acad Sci U S A. 2022 Mar 1;119(9). doi: 10.1073/pnas.2106379119.
High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.
高保真复制冠状病毒（CoVs）的大 RNA 基因组由非结构蛋白 14（nsp14）中的 3'到 5'外切核酸酶（ExoN）介导，该酶切除包括抗病毒药物在内的核苷酸，这些核苷酸是由低保真病毒 RNA 依赖性 RNA 聚合酶（RdRp）错误掺入的，并且还与病毒 RNA 重组和对先天免疫的抵抗力有关。在这里，我们确定了严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）ExoN 与其必需辅助因子 nsp10 复合物的 1.6Å分辨率晶体结构。该结构显示了一个高度碱性且凹陷的表面，侧翼是活性位点，包含 nsp14 的几个赖氨酸残基和 nsp10 的 N 末端氨基。建模表明，这个碱性斑块结合到双链 RNA 底物的模板链上，将新生链的 3'端定位在 ExoN 的活性位点中，这一点得到了突变和计算分析的证实。我们还表明，ExoN 活性可以挽救被索非布韦毒害的停滞 RNA 引物，并允许 RdRp 在链终止药物存在的情况下继续其延伸，在 SARS-CoV-2 复制中生化上模拟校对。分子动力学模拟进一步显示了多结构域 nsp14 的显著灵活性，并表明 nsp10 稳定了 ExoN 以结合底物 RNA，从而支持其外切核酸酶活性。我们的 SARS-CoV-2 ExoN-nsp10 复合物的高分辨率结构为开发抗冠状病毒药物或策略以减弱病毒毒力提供了一个平台。
