tetano
Editor, Senior Moderator
J Biol Chem
. 2020 Sep 23;jbc.AC120.015720.
doi: 10.1074/jbc.AC120.015720. Online ahead of print.
Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action
Egor P Tchesnokov[SUP] 1 [/SUP], Calvin J Gordon[SUP] 1 [/SUP], Emma Woolner[SUP] 1 [/SUP], Dana Kocincova[SUP] 1 [/SUP], Jason K Perry[SUP] 2 [/SUP], Joy Y Feng[SUP] 3 [/SUP], Danielle P Porter[SUP] 4 [/SUP], Matthias Gotte[SUP] 5 [/SUP]
Affiliations
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 S861 and the incorporated RDV-TP. With wild type 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 due to a significant clash with A558. The adjacent V557 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.
Keywords: COVID-19; RNA polymerase; RNA virus; Remdesivir; SARS-2; drug development; drug resistance; viral polymerase.
. 2020 Sep 23;jbc.AC120.015720.
doi: 10.1074/jbc.AC120.015720. Online ahead of print.
Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action
Egor P Tchesnokov[SUP] 1 [/SUP], Calvin J Gordon[SUP] 1 [/SUP], Emma Woolner[SUP] 1 [/SUP], Dana Kocincova[SUP] 1 [/SUP], Jason K Perry[SUP] 2 [/SUP], Joy Y Feng[SUP] 3 [/SUP], Danielle P Porter[SUP] 4 [/SUP], Matthias Gotte[SUP] 5 [/SUP]
Affiliations
- PMID: 32967965
- DOI: 10.1074/jbc.AC120.015720
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 S861 and the incorporated RDV-TP. With wild type 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 due to a significant clash with A558. The adjacent V557 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.
Keywords: COVID-19; RNA polymerase; RNA virus; Remdesivir; SARS-2; drug development; drug resistance; viral polymerase.