Abstract
Ensitrelvir is an antiviral drug that specifically targets the conserved main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, mutations in Mpro 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 Mpro G23 deletion (G23del) with high resistance ( 1,000-fold) to ensitrelvir, but not to the Mpro 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 Mpro (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 Mpro. 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 Mpro that restored viral fitness loss caused by G23del without altering resistance to ensitrelvir. Our results demonstrate that G23del in Mpro can confer resistance to ensitrelvir. Positively, G23del in Mpro does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating SARS-CoV-2 infections containing G23del Mpro 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 Mpro, 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 Mpro (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.