SARS-CoV-2 3CLpro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

Emmanuel Heilmann, Francesco Costacurta, Seyed Arad Moghadasi, Chengjin Ye, Matteo Pavan, Davide Bassani, Andre Volland, Claudia Ascher, Alexander Kurt Hermann Weiss, David Bante, Reuben S. Harris, Stefano Moro, Bernhard Rupp, Luis Martinez-Sobrido, Dorothee von Laer

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50 Scopus citations


Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CLpro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CLpro, and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CLpro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

Original languageEnglish (US)
Article numbereabq7360
JournalScience translational medicine
Issue number678
StatePublished - Jan 11 2023

ASJC Scopus subject areas

  • General Medicine


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