Abstract
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.
Highlights
Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19)
The emergency use authorization (EUA) was largely based on a randomized clinical trial that showed a significant reduction in the time of recovery of hospitalized individuals diagnosed with coronavirus disease 2019 (COVID-19) [2]
Several recent structural and biochemical studies have demonstrated that the active SARS–CoV-2 RNA-dependent RNA polymerase (RdRp) complex is composed of the three nonstructural proteins nsp7, nsp8, and nsp12 [20,21,22,23,24]
Summary
Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). Reactions were monitored on a 17-mer RNA template that contains a single site of incorporation for RDV-TP at position 9 or i (Fig. 1A). The high efficiency of read-through with increasing NTP concentrations suggests that the newly synthesized copy of the RNA genome contains several RDV residues.
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