{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T01:34:49Z","timestamp":1768527289404,"version":"3.49.0"},"posted":{"date-parts":[[2025,10,7]]},"group-title":"Microbiology","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T00:00:00Z","timestamp":1759795200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","award":["U19 AI171403"],"award-info":[{"award-number":["U19 AI171403"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","award":["R01 AI161363"],"award-info":[{"award-number":["R01 AI161363"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","award":["R01 AI161363-03S1"],"award-info":[{"award-number":["R01 AI161363-03S1"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","award":["5N93021C00014"],"award-info":[{"award-number":["5N93021C00014"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Ensitrelvir is an antiviral drug that specifically targets the main protease (M\n                  <jats:sup>pro<\/jats:sup>\n                  ) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been approved for the treatment of coronavirus disease 2019 (COVID-19) due to the conservation of its target protein which is essential in the viral lifecycle. However, SARS-CoV-2 could introduce mutations in the viral proteins to confer resistance to antivirals. Thus, screening for drug-resistant SARS-CoV-2 mutants and elucidating their resistant mechanisms are critical for guiding the selection of effective antiviral therapies. Here, we utilized a luminescent attenuated SARS-CoV-2 (\u03943a7b-Nluc WT) to safely identify ensitrelvir drug-resistant mutants (DRM-E) without the need of using virulent forms of SARS-CoV-2. We isolated a DRM-E containing a G23 deletion (G23del) in M\n                  <jats:sup>pro<\/jats:sup>\n                  with high resistance (&gt;1,000 fold) to ensitrelvir, but not to the other M\n                  <jats:sup>pro<\/jats:sup>\n                  inhibitor (nirmatrelvir) or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del was confirmed by generating a recombinant luminescent attenuated SARS-CoV-2 containing G23del in the non-structural protein 5 (NSP5) gene (\u03943a7b-Nluc G23del). \u03943a7b-Nluc G23del exhibited significant resistance to ensitrelvir in both cultured cells an in K18 hACE2 transgenic mice. Binding affinity revealed that G23del mutation substantially altered M\n                  <jats:sup>pro<\/jats:sup>\n                  binding affinity for ensitrelvir but not nirmatrelvir. In conclusion, our results demonstrate that G23del in M\n                  <jats:sup>pro<\/jats:sup>\n                  can confer high resistance to ensitrelvir. Positively, G23del in M\n                  <jats:sup>pro<\/jats:sup>\n                  does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating infections with SARS-CoV-2 containing G23del with these other approved antivirals.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>SIGNIFICANCE<\/jats:title>\n                  <jats:p>\n                    The clinical use of SARS-CoV-2 antiviral drugs is increasingly challenged by the emergence of drug-resistant mutants. Thus, there is a pressing need to identify and characterize antiviral escape SARS-CoV-2 variants, particularly for FDA-approved antivirals. Our study addresses this by employing a luminescent attenuated virus platform (\u03943a7b-Nluc WT) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we have identified a G23 deletion (G23del) in SARS-CoV-2 M\n                    <jats:sup>pro<\/jats:sup>\n                    , which mediates resistance to ensitrelvir\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\n                    <jats:italic>in vivo<\/jats:italic>\n                    . Importantly, while G23del was able to confer more than 1,000-fold increased resistance to ensitrelvir, SARS-CoV-2 containing G23del remained sensitive to other M\n                    <jats:sup>pro<\/jats:sup>\n                    (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using \u03943a7b-Nluc to safely identify and characterize drug resistant viruses without the biosafety concern of using virulent SARS-CoV-2 and advance the design of next-generation antiviral drugs.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.09.30.679586","type":"posted-content","created":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T20:50:14Z","timestamp":1759870214000},"source":"Crossref","is-referenced-by-count":0,"title":["Identification and characterization of a SARS-CoV-2 M\n                  <sup>pro<\/sup>\n                  G23 deletion ensitrelvir-resistant mutant"],"prefix":"10.64898","author":[{"given":"Yao","family":"Ma","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1934-9494","authenticated-orcid":false,"given":"Chengjin","family":"Ye","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"R.","family":"Rahisuddin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sara H.","family":"Mahmoud","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anastasija","family":"Cupic","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ahmed Magdy","family":"Khalil","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Esteban","family":"Castro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nathaniel","family":"Jackson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mahmoud","family":"Bayoumi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6372-5007","authenticated-orcid":false,"given":"Yogesh K.","family":"Gupta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adolfo","family":"Garcia-Sastre","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard K","family":"Plemper","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luis","family":"Martinez-Sobrido","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025100910501504000_2025.09.30.679586v1.1","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1016\/j.chembiol.2024.03.008","article-title":"SARS-CoV-2 resistance to monoclonal antibodies and small-molecule drugs","volume":"31","year":"2024","journal-title":"Cell Chem Biol"},{"key":"2025100910501504000_2025.09.30.679586v1.2","doi-asserted-by":"crossref","unstructured":"Davies NG , Abbott S , Barnard RC , Jarvis CI , Kucharski AJ , Munday JD , Pearson CAB , Russell TW , Tully DC , Washburne AD , Wenseleers T , Gimma A , Waites W , Wong KLM , van Zandvoort K , Silverman JD , Group CC-W , Consortium C-GU , Diaz-Ordaz K , Keogh R , Eggo RM , Funk S , Jit M , Atkins KE , Edmunds WJ. 2021. Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. 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The rising SARS-CoV-2 JN.1 variant: evolution, infectivity, immune escape, and response strategies. MedComm (2020) 5:e675.","DOI":"10.1002\/mco2.675"},{"key":"2025100910501504000_2025.09.30.679586v1.11","doi-asserted-by":"crossref","first-page":"2193","DOI":"10.1016\/S0140-6736(22)01938-9","article-title":"WHO\u2019s Therapeutics and COVID-19 Living Guideline on mAbs needs to be reassessed","volume":"400","year":"2022","journal-title":"Lancet"},{"key":"2025100910501504000_2025.09.30.679586v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2022.05.001"},{"key":"2025100910501504000_2025.09.30.679586v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-04385-3"},{"key":"2025100910501504000_2025.09.30.679586v1.14","doi-asserted-by":"publisher","DOI":"10.1039\/C4NP00085D"},{"key":"2025100910501504000_2025.09.30.679586v1.15","doi-asserted-by":"crossref","unstructured":"Chan AP , Choi Y , Schork NJ. 2020. Conserved Genomic Terminals of SARS- CoV-2 as Coevolving Functional Elements and Potential Therapeutic Targets. mSphere 5.","DOI":"10.1128\/mSphere.00754-20"},{"key":"2025100910501504000_2025.09.30.679586v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-29104-y"},{"key":"2025100910501504000_2025.09.30.679586v1.17","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06609-0"},{"key":"2025100910501504000_2025.09.30.679586v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-40018-1"},{"key":"2025100910501504000_2025.09.30.679586v1.19","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1011231"},{"key":"2025100910501504000_2025.09.30.679586v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-3527(06)66005-3"},{"key":"2025100910501504000_2025.09.30.679586v1.21","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2147"},{"key":"2025100910501504000_2025.09.30.679586v1.22","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M115.651463"},{"key":"2025100910501504000_2025.09.30.679586v1.23","doi-asserted-by":"publisher","DOI":"10.1126\/science.abl478"},{"key":"2025100910501504000_2025.09.30.679586v1.24","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.2c00117"},{"key":"2025100910501504000_2025.09.30.679586v1.25","doi-asserted-by":"crossref","first-page":"694","DOI":"10.1038\/s42003-023-05071-y","article-title":"Molecular mechanism of ensitrelvir inhibiting SARS-CoV-2 main protease and its variants","volume":"6","year":"2023","journal-title":"Commun Biol"},{"key":"2025100910501504000_2025.09.30.679586v1.26","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkad027"},{"key":"2025100910501504000_2025.09.30.679586v1.27","doi-asserted-by":"crossref","unstructured":"Sasaki M , Tabata K , Kishimoto M , Itakura Y , Kobayashi H , Ariizumi T , Uemura K , Toba S , Kusakabe S , Maruyama Y , Iida S , Nakajima N , Suzuki T , Yoshida S , Nobori H , Sanaki T , Kato T , Shishido T , Hall WW , Orba Y , Sato A , Sawa H . 2023. S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and ameliorates COVID-19 severity in hamsters. 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Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors. 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Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system. Sci Adv 8:eadd7197.","DOI":"10.1126\/sciadv.add7197"},{"key":"2025100910501504000_2025.09.30.679586v1.48","doi-asserted-by":"publisher","DOI":"10.1016\/j.antiviral.2023.105671"},{"key":"2025100910501504000_2025.09.30.679586v1.49","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciad494"},{"key":"2025100910501504000_2025.09.30.679586v1.50","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1016\/j.medj.2023.08.001","article-title":"Multidrug- resistant mutations to antiviral and antibody therapy in an immunocompromised patient infected with SARS-CoV-2","volume":"4","year":"2023","journal-title":"Med"},{"key":"2025100910501504000_2025.09.30.679586v1.51","doi-asserted-by":"crossref","unstructured":"Jochmans D , Liu C , Donckers K , Stoycheva A , Boland S , Stevens SK , De Vita C , Vanmechelen B , Maes P , Trueb B , Ebert N , Thiel V , De Jonghe S , Vangeel L , Bardiot D , Jekle A , Blatt LM , Beigelman L , Symons JA , Raboisson P , Chaltin P , Marchand A , Neyts J , Deval J , Vandyck K. 2023. The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir. mBio 14:e0281522.","DOI":"10.1128\/mbio.02815-22"},{"key":"2025100910501504000_2025.09.30.679586v1.52","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1186\/s12879-023-08797-6","article-title":"Clinical experience of treatment of immunocompromised individuals with persistent SARS-CoV-2 infection based on drug resistance mutations determined by genomic analysis: a descriptive study","volume":"23","year":"2023","journal-title":"BMC Infect Dis"},{"key":"2025100910501504000_2025.09.30.679586v1.53","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.2c01005"},{"key":"2025100910501504000_2025.09.30.679586v1.54","doi-asserted-by":"crossref","unstructured":"Citarella A , Dimasi A , Moi D , Passarella D , Scala A , Piperno A , Micale N . 2023. Recent Advances in SARS-CoV-2 Main Protease Inhibitors: From Nirmatrelvir to Future Perspectives. Biomolecules 13.","DOI":"10.20944\/preprints202308.0055.v1"},{"key":"2025100910501504000_2025.09.30.679586v1.55","doi-asserted-by":"publisher","DOI":"10.1002\/cpmc.126"},{"key":"2025100910501504000_2025.09.30.679586v1.56","doi-asserted-by":"crossref","unstructured":"Ye C , Chiem K , Park JG , Oladunni F , Platt RN , 2nd, Anderson T , Almazan F , de la Torre JC , Martinez-Sobrido L. 2020. Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome. mBio 11.","DOI":"10.1128\/mBio.02168-20"},{"key":"2025100910501504000_2025.09.30.679586v1.57","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-021-03819-2"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.30.679586","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:44:52Z","timestamp":1768470292000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.30.679586"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,7]]},"references-count":57,"URL":"https:\/\/doi.org\/10.1101\/2025.09.30.679586","relation":{},"subject":[],"published":{"date-parts":[[2025,10,7]]},"subtype":"preprint"}}