{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T22:27:10Z","timestamp":1788992830244,"version":"build-2803163510"},"reference-count":57,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T00:00:00Z","timestamp":1783468800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T00:00:00Z","timestamp":1783468800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2026,7,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Ensitrelvir is an antiviral drug that specifically targets the conserved main protease (M\n                      <jats:sup>pro<\/jats:sup>\n                      ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, mutations in M\n                      <jats:sup>pro<\/jats:sup>\n                      could confer resistance to antivirals, including ensitrelvir. Thus, identifying SARS-CoV-2 drug-resistant mutants and elucidating their mechanisms of resistance are critical for guiding the selection of effective antiviral therapies. Here, we utilized a recombinant luminescent attenuated SARS-CoV-2 lacking the open reading frames (ORF) 3a and 7b proteins (\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 M\n                      <jats:sup>pro<\/jats:sup>\n                      G23 deletion (G23del) with high resistance (~1,000-fold) to ensitrelvir, but not to the M\n                      <jats:sup>pro<\/jats:sup>\n                      inhibitor nirmatrelvir or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del in ensitrelvir resistance was confirmed by generating a \u03943a7b-Nluc containing G23del in M\n                      <jats:sup>pro<\/jats:sup>\n                      (\u03943a7b-Nluc G23del). \u03943a7b-Nluc G23del exhibited resistance to ensitrelvir in both cultured cells and in K18 hACE2 transgenic mice. Binding affinity revealed that the G23del mutation altered ensitrelvir, but not nirmatrelvir, binding to M\n                      <jats:sup>pro<\/jats:sup>\n                      . Notably, while \u03943a7b-Nluc G23del was affected in viral fitness, serial passage of \u03943a7b-Nluc G23del in the absence of ensitrelvir resulted in the emergence of substitution L50F in M\n                      <jats:sup>pro<\/jats:sup>\n                      that restored viral fitness loss caused by G23del without altering resistance to ensitrelvir. Our results demonstrate that G23del in M\n                      <jats:sup>pro<\/jats:sup>\n                      can confer 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 SARS-CoV-2 infections containing G23del M\n                      <jats:sup>pro<\/jats:sup>\n                      with other approved antivirals.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        The clinical use of severe acute respiratory syndrome coronavirus 2 (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 Food and Drug Administration-approved antivirals. Our study addresses this by employing a luminescent attenuated SARS-CoV-2 platform (\u03943a7b-Nluc wild type [WT]) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we 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 toggle=\"yes\">in vitro<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        . Importantly, while G23del was able to confer ~1,000-fold increased resistance to ensitrelvir, a \u03943a7b-Nluc 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 WT to safely identify and characterize drug-resistant viruses without the biosafety concern of using virulent WT SARS-CoV-2 and advance the design of next-generation antiviral drugs.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00584-26","type":"journal-article","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T13:01:19Z","timestamp":1780491679000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Identification and characterization of a SARS-CoV-2 M\n                    <sup>pro<\/sup>\n                    G23 deletion ensitrelvir-resistant mutant"],"prefix":"10.1128","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0815-7910","authenticated-orcid":false,"given":"Yao","family":"Ma","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00wbskb04","id-type":"ROR","asserted-by":"publisher"}],"name":"Texas Biomedical Research Institute","place":["San Antonio, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1934-9494","authenticated-orcid":true,"given":"Chengjin","family":"Ye","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00wbskb04","id-type":"ROR","asserted-by":"publisher"}],"name":"Texas Biomedical Research Institute","place":["San Antonio, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R.","family":"Rahisuddin","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02f6dcw23","id-type":"ROR","asserted-by":"publisher"}],"name":"Greehey Children\u2019s Cancer Research Institute, 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