Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.
Raw sequencing results for passaging in Huh7-ACE2 cells. Samples are denoted as passage number, followed by well number. Cut site mutations are denoted as “cs”.
Passage transitions used for construction of the pathway analysis in Figure 3a.
GenBank Accession IDs for sequences from passaging in Vero E6 cells.
Oligos used for next-generation sequencing.
SRA Accession IDs for raw sequencing data from passaging in Huh7-ACE2 cells.
Oligos used for site-directed mutagenesis to produce isogenic recombinant SARS-CoV-2.
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Iketani, S., Mohri, H., Culbertson, B. et al. Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir. Nature (2022). https://doi.org/10.1038/s41586-022-05514-2