Tchesnokov Egor P, Gordon Calvin J, Woolner Emma, Kocinkova Dana, Perry Jason K, Feng Joy Y, Porter Danielle P, Götte Matthias
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
Gilead Sciences, Inc., Foster City, California, USA.
J Biol Chem. 2020 Nov 20;295(47):16156-16165. doi: 10.1074/jbc.AC120.015720. Epub 2020 Sep 23.
Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). RDV targets the viral RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have previously shown that incorporation of the active triphosphate form of RDV (RDV-TP) at position i causes delayed chain termination at position i + 3. Here we demonstrate that the S861G mutation in RdRp eliminates chain termination, which confirms the existence of a steric clash between Ser-861 and the incorporated RDV-TP. With WT RdRp, increasing concentrations of NTP pools cause a gradual decrease in termination and the resulting read-through increases full-length product formation. Hence, RDV residues could be embedded in copies of the first RNA strand that is later used as a template. We show that the efficiency of incorporation of the complementary UTP opposite template RDV is compromised, providing a second opportunity to inhibit replication. A structural model suggests that RDV, when serving as the template for the incoming UTP, is not properly positioned because of a significant clash with Ala-558. The adjacent Val-557 is in direct contact with the template base, and the V557L mutation is implicated in low-level resistance to RDV. We further show that the V557L mutation in RdRp lowers the nucleotide concentration required to bypass this template-dependent inhibition. The collective data provide strong evidence to show that template-dependent inhibition of SARS-CoV-2 RdRp by RDV is biologically relevant.
瑞德西韦（RDV）是一种直接作用的抗病毒药物，用于治疗重症2019冠状病毒病（COVID-19）患者。RDV靶向严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的病毒RNA依赖性RNA聚合酶（RdRp）。我们之前已经表明，在第i位掺入活性三磷酸形式的RDV（RDV-TP）会导致在第i + 3位延迟链终止。在此我们证明，RdRp中的S861G突变消除了链终止，这证实了Ser-861与掺入的RDV-TP之间存在空间冲突。对于野生型RdRp，增加NTP池的浓度会导致终止逐渐减少，由此产生的通读增加了全长产物的形成。因此，RDV残基可能嵌入到随后用作模板的第一条RNA链的拷贝中。我们表明，与模板RDV相对的互补UTP的掺入效率受到损害，这提供了第二个抑制复制的机会。一个结构模型表明，当RDV作为进入的UTP的模板时，由于与Ala-558发生严重冲突而未正确定位。相邻的Val-557与模板碱基直接接触，V557L突变与对RDV的低水平抗性有关。我们进一步表明，RdRp中的V557L突变降低了绕过这种模板依赖性抑制所需的核苷酸浓度。这些综合数据提供了有力证据，表明RDV对SARS-CoV-2 RdRp的模板依赖性抑制具有生物学相关性。
