Abstract: S C I E N C E T R A N S L AT I O N A L M E D I C I N E | R E S E A R C H A R T I C L E C O R O N AV I R U S SARS-CoV-2 3CLpro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376 Emmanuel Heilmann1†*, Francesco Costacurta1†, Seyed Arad Moghadasi2, Chengjin Ye3, Matteo Pavan4, Davide Bassani4, Andre Volland1, Claudia Ascher5, Alexander Kurt Hermann Weiss5, David Bante1, Reuben S. Harris2,6,7, Stefano Moro4, Bernhard Rupp8,9, Luis Martinez-Sobrido3, Dorothee von Laer1* Copyright © 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). 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 SARSCoV-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. In late 2019, the zoonotic transmission of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), into the human population (1) led to worldwide efforts to find effective treatments against the various pathologies caused by the virus. Inhibitors of viral enzymes, such as proteases, have proven to be highly potent drugs in the treatment of HIV and hepatitis C virus infections. However, resistant viruses rapidly emerge unless the protease inhibitors are given in combination with other directly acting antivirals (2, 3). SARS-CoV-2 encodes two proteases. The 3-chymotrypsin–like protease (3CLpro) cleaves 11 sites in the viral polyproteins pp1a and pp1ab and is also referred to as the main protease or nonstructural protein 5, indicating that it cleaves more sites than the second protease and its location within the polyproteins, respectively (4). The second viral protease, papain-like protease (PLpro), cleaves three additional sites in pp1a and pp1ab (5). Thus, both 1 Institute of Virology, Medical University of Innsbruck, Innsbruck, 6020, Austria. Department of Biochemistry, Molecular Biology and Biophysics, Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA. 3Texas Biomedical Research Institute, San Antonio, TX 78229, USA. 4Molecular Modeling Section (MMS), Department of..