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. 2022 Jul 19;66(7):e0019822.
doi: 10.1128/aac.00198-22. Epub 2022 Jun 16.

In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance

Liva Checkmahomed  1 , Julie Carbonneau  1 , Venice Du Pont  2 , Nicholas C Riola  2 , Jason K Perry  2 , Jiani Li  2 , Bastien Paré  3 , Shawn M Simpson  3 , Martin A Smith  3 , Danielle P Porter  2 , Guy Boivin  1
Affiliations

In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance

Liva Checkmahomed et al. Antimicrob Agents Chemother. .

Abstract

In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 μM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC50). When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to remdesivir resistance.

Keywords: Nsp12 polymerase; SARS-CoV-2; remdesivir; resistance.

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Conflict of interest statement

The authors declare a conflict of interest. 5 of the authors work at Gilead Sciences.

Figures

FIG 1
FIG 1
Antiviral assays and replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-Nluc viruses. (A) Activity of remdesivir (RDV) against SARS-CoV-2-Nluc WA1 expressing Nsp12-WT, Nsp12-P323L, and RDV-selected substitution Nsp12-V166L/P323L in A549-hACE2 cells infected at MOI 0.05. Dose-response curves are based on at least 2 independent experiments with two technical replicates each and fit based on a nonlinear regression model using GraphPad Prism 8. (B) Viral replication kinetics of SARS-CoV-2-Nluc WA1 expressing Nsp12-WT, Nsp12-P323L, and RDV-selected substitution Nsp12-V166L/P323L in A549-hACE2-TMPRSS2 cells infected at multiplicity of infection of 0.01. Viral titer for each indicated time point was determined by plaque-reduction assays performed in Vero-TMPRSS2 cells in triplicate. EC50, 50% effective concentration. Statistical analysis was performed by two-way ANOVA with Tukey’s post hoc comparison tests. (NS, not significant; ****, P ≤ 0.0001).
FIG 2
FIG 2
Model of preincorporated RDV triphosphate (RDV-TP) with a V166L substitution (magenta) in SARS-CoV-2 Nsp12 (green). V166L is outside of the active site but contacts Motif A (cyan) and Motif D (pink) residues. Nsp7 and Nsp8 are visible in white and yellow, respectively. The nascent RNA strand is in red and the template strand is in blue. The software Maestro, Prime, and Macromodel, within the Schrödinger software suite, were used to perform structural analysis.
See this image and copyright information in PMC

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