tetano
Editor, Senior Moderator
mBio
. 2026 Jun 3:e0058426.
doi: 10.1128/mbio.00584-26. Online ahead of print.
Identification and characterization of a SARS-CoV-2 M[SUP]pro[/SUP] G23 deletion ensitrelvir-resistant mutant
Yao Ma[SUP] #[/SUP][SUP] 1 [/SUP], Chengjin Ye[SUP] #[/SUP][SUP] 1 [/SUP], R Rahisuddin[SUP] 2 3 [/SUP], Sara H Mahmoud[SUP] 1 4 [/SUP], Anastasija Cupic[SUP] 5 6 [/SUP], Ahmed Magdy Khalil[SUP] 1 7 [/SUP], Esteban Castro[SUP] 1 [/SUP], Nathaniel Jackson[SUP] 1 [/SUP], Mahmoud Bayoumi[SUP] 1 8 [/SUP], Shailee Arya[SUP] 2 3 [/SUP], Yogesh K Gupta[SUP] 2 3 [/SUP], Adolfo García-Sastre[SUP] 6 9 10 11 12 13 [/SUP], Richard K Plemper[SUP] 14 [/SUP], Luis Martinez-Sobrido[SUP] 1 [/SUP]
Affiliations
Ensitrelvir is an antiviral drug that specifically targets the conserved main protease (M[SUP]pro[/SUP]) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, mutations in M[SUP]pro[/SUP] 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 (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 M[SUP]pro[/SUP] G23 deletion (G23del) with high resistance (~1,000-fold) to ensitrelvir, but not to the M[SUP]pro[/SUP] inhibitor nirmatrelvir or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del in ensitrelvir resistance was confirmed by generating a Δ3a7b-Nluc containing G23del in M[SUP]pro[/SUP] (Δ3a7b-Nluc G23del). Δ3a7b-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[SUP]pro[/SUP]. Notably, while Δ3a7b-Nluc G23del was affected in viral fitness, serial passage of Δ3a7b-Nluc G23del in the absence of ensitrelvir resulted in the emergence of substitution L50F in M[SUP]pro[/SUP] that restored viral fitness loss caused by G23del without altering resistance to ensitrelvir. Our results demonstrate that G23del in M[SUP]pro[/SUP] can confer resistance to ensitrelvir. Positively, G23del in M[SUP]pro[/SUP] does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating SARS-CoV-2 infections containing G23del M[SUP]pro[/SUP] with other approved antivirals.IMPORTANCEThe 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 (Δ3a7b-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[SUP]pro[/SUP], which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer ~1,000-fold increased resistance to ensitrelvir, a Δ3a7b-Nluc containing G23del remained sensitive to other M[SUP]pro[/SUP] (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using Δ3a7b-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.
Keywords: G23 deletion; SARS-CoV-2; attenuated virus; drug resistance mutations; ensitrelvir.
. 2026 Jun 3:e0058426.
doi: 10.1128/mbio.00584-26. Online ahead of print.
Identification and characterization of a SARS-CoV-2 M[SUP]pro[/SUP] G23 deletion ensitrelvir-resistant mutant
Yao Ma[SUP] #[/SUP][SUP] 1 [/SUP], Chengjin Ye[SUP] #[/SUP][SUP] 1 [/SUP], R Rahisuddin[SUP] 2 3 [/SUP], Sara H Mahmoud[SUP] 1 4 [/SUP], Anastasija Cupic[SUP] 5 6 [/SUP], Ahmed Magdy Khalil[SUP] 1 7 [/SUP], Esteban Castro[SUP] 1 [/SUP], Nathaniel Jackson[SUP] 1 [/SUP], Mahmoud Bayoumi[SUP] 1 8 [/SUP], Shailee Arya[SUP] 2 3 [/SUP], Yogesh K Gupta[SUP] 2 3 [/SUP], Adolfo García-Sastre[SUP] 6 9 10 11 12 13 [/SUP], Richard K Plemper[SUP] 14 [/SUP], Luis Martinez-Sobrido[SUP] 1 [/SUP]
Affiliations
- PMID: 42233671
- DOI: 10.1128/mbio.00584-26
Ensitrelvir is an antiviral drug that specifically targets the conserved main protease (M[SUP]pro[/SUP]) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, mutations in M[SUP]pro[/SUP] 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 (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 M[SUP]pro[/SUP] G23 deletion (G23del) with high resistance (~1,000-fold) to ensitrelvir, but not to the M[SUP]pro[/SUP] inhibitor nirmatrelvir or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del in ensitrelvir resistance was confirmed by generating a Δ3a7b-Nluc containing G23del in M[SUP]pro[/SUP] (Δ3a7b-Nluc G23del). Δ3a7b-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[SUP]pro[/SUP]. Notably, while Δ3a7b-Nluc G23del was affected in viral fitness, serial passage of Δ3a7b-Nluc G23del in the absence of ensitrelvir resulted in the emergence of substitution L50F in M[SUP]pro[/SUP] that restored viral fitness loss caused by G23del without altering resistance to ensitrelvir. Our results demonstrate that G23del in M[SUP]pro[/SUP] can confer resistance to ensitrelvir. Positively, G23del in M[SUP]pro[/SUP] does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating SARS-CoV-2 infections containing G23del M[SUP]pro[/SUP] with other approved antivirals.IMPORTANCEThe 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 (Δ3a7b-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[SUP]pro[/SUP], which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer ~1,000-fold increased resistance to ensitrelvir, a Δ3a7b-Nluc containing G23del remained sensitive to other M[SUP]pro[/SUP] (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using Δ3a7b-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.
Keywords: G23 deletion; SARS-CoV-2; attenuated virus; drug resistance mutations; ensitrelvir.