{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T02:35:00Z","timestamp":1789180500809,"version":"build-2803163510"},"reference-count":65,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2023,9,21]],"date-time":"2023-09-21T00:00:00Z","timestamp":1695254400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Candys Foundation"},{"name":"China Scholarship Council"},{"name":"Amager and Hvidovre Hospital Research Foundation"},{"name":"Danish Agency for Science and Higher Education"},{"name":"Independent Research Fund Denmark"},{"name":"Novo Nordisk Foundation including a Distinguished Investigator Grant"},{"name":"Mauritzen la Fontaine Fonden"},{"name":"Mauritzen La Fontaine Familiefond"},{"name":"Region H Foundation"},{"name":"Toyota Foundation"},{"name":"Weimann Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration\u2013response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration\u2013response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.<\/jats:p>","DOI":"10.3390\/v15091970","type":"journal-article","created":{"date-parts":[[2023,9,21]],"date-time":"2023-09-21T21:08:36Z","timestamp":1695330516000},"page":"1970","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Substitutions in SARS-CoV-2 Mpro Selected by Protease Inhibitor Boceprevir Confer Resistance to Nirmatrelvir"],"prefix":"10.3390","volume":"15","author":[{"given":"Karen Anbro","family":"Gammeltoft","sequence":"first","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6330-3170","authenticated-orcid":false,"given":"Yuyong","family":"Zhou","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Line Abildgaard","family":"Ryberg","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Long V.","family":"Pham","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alekxander","family":"Binderup","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos Rene Duarte","family":"Hernandez","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7748-2840","authenticated-orcid":false,"given":"Anna","family":"Offersgaard","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulrik","family":"Fahn\u00f8e","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9754-2663","authenticated-orcid":false,"given":"G\u00fcnther Herbert Johannes","family":"Peters","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Santseharay","family":"Ramirez","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7815-4806","authenticated-orcid":false,"given":"Jens","family":"Bukh","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2805-0256","authenticated-orcid":false,"given":"Judith Margarete","family":"Gottwein","sequence":"additional","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Ketteg\u00e5rd Alle 30, 2650 Hvidovre, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,21]]},"reference":[{"key":"ref_1","unstructured":"(2023, June 28). 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