biostudies-literatureDeschenes NMNational Institute of Allergy and Infectious DiseasesGovernment of SaskatchewanNIAID NIH HHSVancouver General Hospital FoundationNatural Sciences and Engineering Research CouncilNational Research CouncilCanada Foundation for InnovationNational Institutes of HealthUniversity of SaskatchewanNIH HHSCanadian Institutes of Health ResearchCIHRe789-e798https://www.ebi.ac.uk/biostudies/studies/S-EPMC12614980biostudies-literatureUnknown232(5)<h4>Background</h4>The main protease (Mpro) is one of the most attractive targets for antiviral drug discovery against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Mutations in Mpro have been linked to resistance against nirmatrelvir-ritonavir (NIR-RIT), an important therapy for SARS-CoV-2 infection. This study aimed to identify low-frequency antiviral resistance mutations in Mpro from NIR-RIT-treated patients and to analyze the enzymatic properties, inhibitor susceptibility, and structural features of new Mpro clinical variants.<h4>Methods</h4>We screened 1528 SARS-CoV-2-positive patients from 2 hospitals and identified 17 who remained positive after treatment. Whole-genome sequencing of nasopharyngeal specimens was conducted to identify Mpro clinical variants. The impactThe Journal of infectious diseasesFunctional and Structural Characterization of Treatment-Emergent Nirmatrelvir Resistance Mutations at Low Frequencies in the Main Protease (Mpro) Reveals a Unique Evolutionary Route for SARS-CoV-2 to Gain Resistance.PMC12614980R44 AI1651881R44AI165188-01A1Worrall LJLi AXLefebvre MWaglechner NMaguire FFarooqi LThomas MDeschenes NMStrynadka NCJYoung RNMcGeer AMalik IZhong ZSiddiqi LPaetzel MKozak RASmith JRJean FMoinuddin AAftanas PCherkasov APerez-Vargas JMosimann WALim JKenward CfalseFunctional and Structural Characterization of Treatment-Emergent Nirmatrelvir Resistance Mutations at Low Frequencies in the Main Protease (Mpro) Reveals a Unique Evolutionary Route for SARS-CoV-2 to Gain Resistance.<h4>Background</h4>The main protease (Mpro) is one of the most attractive targets for antiviral drug discovery against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Mutations in Mpro have been linked to resistance against nirmatrelvir-ritonavir (NIR-RIT), an important therapy for SARS-CoV-2 infection. This study aimed to identify low-frequency antiviral resistance mutations in Mpro from NIR-RIT-treated patients and to analyze the enzymatic properties, inhibitor susceptibility, and structural features of new Mpro clinical variants.<h4>Methods</h4>We screened 1528 SARS-CoV-2-positive patients from 2 hospitals and identified 17 who remained positive after treatment. Whole-genome sequencing of nasopharyngeal specimens was conducted to identify Mpro clinical variants. The impact2025-01-01T00:00:00Z2025 Nov2026-06-06T13:30:49.954Z2026-05-31T03:07:48.661ZS-EPMC126149804045923310.1093/infdis/jiaf294