{"doi":"10.1101/2025.09.30.679586","title":"Identification and characterization of a SARS-CoV-2 M <sup>pro</sup> G23 deletion ensitrelvir-resistant mutant","abstract":"ABSTRACT Ensitrelvir is an antiviral drug that specifically targets the main protease (M pro ) 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 (Δ3a7b-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 pro with high resistance (&gt;1,000 fold) to ensitrelvir, but not to the other M pro 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 (Δ3a7b-Nluc G23del). Δ3a7b-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 pro binding affinity for ensitrelvir but not nirmatrelvir. In conclusion, our results demonstrate that G23del in M pro can confer high resistance to ensitrelvir. Positively, G23del in M pro 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. SIGNIFICANCE 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 (Δ3a7b-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 pro , which mediates resistance to ensitrelvir in vitro and in vivo . 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 pro (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using Δ3a7b-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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":577119,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9476,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":2361,"name":"Chengjin Ye","orcid":"0000-0002-1934-9494","position":1,"is_corresponding":false},{"id":1446040,"name":"R Rahisuddin","orcid":null,"position":2,"is_corresponding":false},{"id":1486679,"name":"Sarah Mahmoud","orcid":null,"position":3,"is_corresponding":false},{"id":2417,"name":"Anastasija Čupić","orcid":"0000-0002-1291-1042","position":4,"is_corresponding":false},{"id":1021518,"name":"Ahmed Magdy Khalil","orcid":"0000-0002-1358-6755","position":5,"is_corresponding":false},{"id":1220895,"name":"Esmeralda Gracián-Castro","orcid":"0000-0002-0850-2465","position":6,"is_corresponding":false},{"id":651951,"name":"Nathaniel Jackson","orcid":"0000-0002-9654-6926","position":7,"is_corresponding":false},{"id":1220896,"name":"Mahmoud Bayoumi","orcid":"0000-0002-1265-3047","position":8,"is_corresponding":false},{"id":240061,"name":"Yogesh K. Gupta","orcid":"0000-0001-6372-5007","position":9,"is_corresponding":false},{"id":130447,"name":"Adolfo Garcia-Sastre","orcid":null,"position":10,"is_corresponding":false},{"id":228962,"name":"Richard K. Plemper","orcid":"0000-0003-2034-2107","position":11,"is_corresponding":false},{"id":2423,"name":"Luis Martínez‐Sobrido","orcid":"0000-0001-7084-0804","position":12,"is_corresponding":false},{"id":520765,"name":"Yao Ma","orcid":"0000-0002-9061-3999","position":0,"is_corresponding":true}],"reference_count":55,"raw_metadata":null,"created_at":"2026-07-19T02:58:04.622308Z","pmid":"41279697","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}