SARS-CoV-2 Remdesivir Exposure Leads to Different Evolutionary Pathways That Converge in Moderate Levels of Drug Resistance

Various SARS-CoV-2 remdesivir resistance-associated substitutions (RAS) have been reported, but a comprehensive comparison of their resistance levels is lacking. We identified novel RAS and performed head-to-head comparisons with known RAS in Vero E6 cells. A remdesivir escape polyclonal virus exhibited a 3.6-fold increase in remdesivir EC50 and mutations throughout the genome, including substitutions in nsp12 (E796D) and nsp14 (A255S). However, in reverse-genetics infectious assays, viruses harboring both these substitutions exhibited only a slight decrease in remdesivir susceptibility (1.3-fold increase in EC50). The nsp12-E796D substitution did not impair viral fitness (Vero E6 cells or Syrian hamsters) and was reported in a remdesivir-treated COVID-19 patient. In replication assays, a subgenomic replicon containing nsp12-E796D+nsp14-A255S led to a 16.1-fold increase in replication under remdesivir treatment. A comparison with known RAS showed that S759A, located in the active site of nsp12, conferred the highest remdesivir resistance (106.1-fold increase in replication). Nsp12-RAS V166A/L, V792I, E796D or C799F, all adjacent to the active site, caused intermediate resistance (2.0- to 11.5-fold), whereas N198S, D484Y, or E802D, located farther from the active site, showed no resistance (≤2.0-fold). In conclusion, our classification system, correlating replication under remdesivir treatment with RAS location in nsp12, shows that most nsp12-RAS cause moderate resistance.