Min, Seong Cheol Seo, Jin-Ju Jeong, Ju Hwan Kim, Beom Kyu Park, Ji-Hyun Lee, Ju Ryeong Lee, Dong Gyu Lee, Gi Chan An, Se Hee Baek, Yun Hee Choi, Young Ki Choo, Hyunah Park, Hyo Yong Kim, Gyeongmin Jeon, Byungsun Shin, Sang Chul Song, Min-Suk 2025-12-02T09:00:48Z 2025-12-02T09:00:48Z 2025-11-24 2025-11 https://pubs.kist.re.kr/handle/201004/153726 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 (Mpro), their distinct susceptibility profiles may allow alternative therapeutic approaches. Here, we identify a deletion mutation at glycine 23 (Δ23G) in Mpro 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 Mpro-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 Mpro inhibitors, supporting potential sequential or alternative use of nirmatrelvir and ensitrelvir in patients requiring prolonged antiviral treatment. English Nature Publishing Group A SARS-CoV-2 Mpro mutation conferring ensitrelvir resistance paradoxically increases nirmatrelvir susceptibility Article 10.1038/s41467-025-65767-z 1 Nature Communications, v.16 Nature Communications 16 Y scie scopus 001629196400001 Multidisciplinary Sciences Science & Technology - Other Topics Article