Identification and characterization of a SARS-CoV-2 M^pro G23 deletion ensitrelvir-resistant mutant

Ensitrelvir is an antiviral drug that specifically targets the conserved main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, mutations in M^pro could confer resistance to antivirals, including ensitrelvir. Thus, identifying SARS-CoV-2 drug-resistant mutants and elucidating their mechanisms of resistance are critical for guiding the selection of effective antiviral therapies. Here, we utilized a recombinant luminescent attenuated SARS-CoV-2 lacking the open reading frames (ORF) 3a and 7b proteins (Δ3a7b-Nluc WT) to safely identify ensitrelvir drug-resistant mutants (DRM-E) without the need of using virulent forms of SARS-CoV-2. We isolated a DRM-E containing a M^pro G23 deletion (G23del) with high resistance (~1,000-fold) to ensitrelvir, but not to the M^pro inhibitor nirmatrelvir or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del in ensitrelvir resistance was confirmed by generating a Δ3a7b-Nluc containing G23del in M^pro (Δ3a7b-Nluc G23del). Δ3a7b-Nluc G23del exhibited resistance to ensitrelvir in both cultured cells and in K18 hACE2 transgenic mice. Binding affinity revealed that the G23del mutation altered ensitrelvir, but not nirmatrelvir, binding to M^pro. Notably, while Δ3a7b-Nluc G23del was affected in viral fitness, serial passage of Δ3a7b-Nluc G23del in the absence of ensitrelvir resulted in the emergence of substitution L50F in M^pro that restored viral fitness loss caused by G23del without altering resistance to ensitrelvir. Our results demonstrate that G23del in M^pro can confer resistance to ensitrelvir. Positively, G23del in M^pro does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating SARS-CoV-2 infections containing G23del M^pro with other approved antivirals.IMPORTANCEThe clinical use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral drugs is increasingly challenged by the emergence of drug-resistant mutants. Thus, there is a pressing need to identify and characterize antiviral escape SARS-CoV-2 variants, particularly for Food and Drug Administration-approved antivirals. Our study addresses this by employing a luminescent attenuated SARS-CoV-2 platform (Δ3a7b-Nluc wild type [WT]) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we identified a G23 deletion (G23del) in SARS-CoV-2 M^pro, which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer ~1,000-fold increased resistance to ensitrelvir, a Δ3a7b-Nluc containing G23del remained sensitive to other M^pro (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using Δ3a7b-Nluc WT to safely identify and characterize drug-resistant viruses without the biosafety concern of using virulent WT SARS-CoV-2 and advance the design of next-generation antiviral drugs.