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.
【저자키워드】 COVID-19, SARS-CoV-2, coronavirus, Drug development, Remdesivir, Replication, RdRP, RNA-dependent RNA polymerase, CoV, drug resistance, RNA polymerase, RNA virus, SARS-2, delayed chain termination, viral polymerase, 【초록키워드】 Mutation, Antiviral, RNA, Patient, target, RDV, Evidence, nucleotide, Concentration, Contact, Efficiency, complementary, acute respiratory syndrome, severe coronavirus disease, residue, treat, Lower, full-length, direct-acting, opposite, decrease, shown, resulting, inhibit, required, increase, cause, implicated, steric, NTP, positioned, UTP, V557L, 【제목키워드】 mechanism of action, reveal,