SARS-CoV-2 3CLpro mutations confer resistance to Paxlovid (nirmatrelvir/ritonavir) in a VSV-based, non-gain-of-function system

Protease inhibitors are among the most powerful antiviral drugs. A first protease inhibitor against the SARS-CoV-2 protease 3CLpro, Paxlovid (nirmatrelvir / ritonavir), has recently been authorized by the U.S. FDA for emergency use (EUA 105 Pfizer Paxlovid). To find resistant mutants against the protease-inhibitor-component of Paxlovid, nirmatrelvir, we engineered a chimeric Vesicular Stomatitis Virus (VSV). By replacing an intergenic region, which is essential for separate gene transcription, with 3CLpro, this chimeric VSV became dependent on the protease to process two of its genes. We then applied selective pressure with nirmatrelvir to induce mutations. The effect of those mutants was confirmed by re-introduction in the 3CLpro and testing with a recently developed cellular assay. Furthermore, we found that mutations predicted by our method already exist in SARS-CoV-2 sequence depositions in NCBI and GISAID data bases. These may represent emerging resistant virus variants or a natural heterogeneity in the susceptibility to nirmatrelvir. One-Sentence SummaryMutations of the main protease of SARS-CoV-2 result in resistance against licensed drugs such as Paxlovid (nirmatrelvir / ritonavir). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/495455v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1d539d3org.highwire.dtl.DTLVardef@1c76534org.highwire.dtl.DTLVardef@1c54c4corg.highwire.dtl.DTLVardef@14355d_HPS_FORMAT_FIGEXP M_FIG C_FIG