期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 47, 页码 16156-16165出版社
ELSEVIER
DOI: 10.1074/jbc.AC120.015720
关键词
COVID-19; coronavirus; CoV; SARS-CoV-2; RNA-dependent RNA polymerase; RdRp; replication; remdesivir; delayed chain termination; viral polymerase; drug development; drug resistance; RNA polymerase; RNA virus; SARS-2
资金
- Canadian Institutes of Health Research (CIHR) [170343]
- Gilead Sciences Grant
- Alberta Ministry of Economic Development, Trade and Tourism by the Major Innovation Fund Program for the AMR-One Health Consortium
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.
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