A SARS-CoV-2 Mpro 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 identified a novel deletion mutation at glycine 23 (Δ23G) in M ^pro that conferred substantial resistance to ensitrelvir (~ 35-fold) while paradoxically increasing susceptibility to nirmatrelvir (~ 8-fold). This opposite susceptibility pattern was confirmed both in vitro and in a hamster infection model. Recombinant viruses carrying M ^pro -Δ23G exhibited impaired replication, pathogenicity, and transmissibility compared to wild-type, though the co-occurring mutation T45I partially restored viral fitness. Structural analyses revealed 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.