A SARS-CoV-2 M^pro mutation conferring ensitrelvir resistance paradoxically increases nirmatrelvir susceptibility

SARS-CoV-2 variants resistant to current antivirals remain a significant threat, particularly in high-risk patients. Although nirmatrelvir and ensitrelvir both target the viral 3CL protease (M^pro), their distinct susceptibility profiles may allow alternative therapeutic approaches. Here, we identify a deletion mutation at glycine 23 (Δ23G) in M^pro that conferred high-level resistance to ensitrelvir ( ~ 35-fold) while paradoxically increasing susceptibility to nirmatrelvir ( ~ 8-fold). This opposite susceptibility pattern is confirmed both in vitro and in a male hamster infection model. Recombinant viruses carrying M^pro-Δ23G exhibit impaired replication, pathogenicity, and transmissibility compared to wild-type, though the co-occurring mutation T45I partially restore viral fitness. Structural analyses reveal critical conformational changes in the catalytic loop (Ile136-Val148) and β-hairpin loop (Cys22-Thr26), directly influencing inhibitor binding selectivity. These results highlight differential resistance profiles of M^pro inhibitors, supporting potential sequential or alternative use of nirmatrelvir and ensitrelvir in patients requiring prolonged antiviral treatment.