Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system
Yuyong Zhou, Karen Anbro Gammeltoft, Abildgaard Ryberg, Long V Pham, Helena Damtoft Tjørnelund, Alekxander Binderup, Carlos Rene Duarte Hernandez, Carlota Fernandez-Antunez, Anna Offersgaard, Ulrik Fahnøe, Günther Herbert, Johannes Peters, Santseharay Ramirez, Jens Bukh, Judith Margarete Gottwein
The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.
Only interactions with differences larger than 5% were included in fig. S6 .
Sarbecovirus Mpro alignment Sarbecovirus Mpro amino acid sequences were aligned by Geneious Prime 2019.2.3 software. SARS-CoV-2 isolate SARS-CoV-2-WuhanHB (GenBank: NC045512) residues Ser 1 -Gln 306 were used as the reference. On the basis of the representative sarbecovirus spike receptor-binding domain amino acid sequences, sarbecoviruses phylogenetically cluster into four clades (49) . Forty-three representative sarbecovirus sequences obtained from GenBank or GISAID from all four clades were chosen for alignment (49) .
Supplementary Materials
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