TY - JOUR

T1 - Substitutions in SARS-CoV-2 Mpro Selected by Protease Inhibitor Boceprevir Confer Resistance to Nirmatrelvir

AU - Gammeltoft, Karen Anbro

AU - Zhou, Yuyong

AU - Ryberg, Line Abildgaard

AU - Pham, Long V.

AU - Binderup, Alekxander

AU - Duarte Hernandez, Carlos Rene

AU - Offersgaard, Anna

AU - Fahnøe, Ulrik

AU - Peters, Günther Herbert Johannes

AU - Ramirez, Santseharay

AU - Bukh, Jens

AU - Gottwein, Judith Margarete

N1 - Publisher Copyright: © 2023 by the authors.

PY - 2023

Y1 - 2023

N2 - Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration–response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration–response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.

AB - Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration–response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration–response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.

KW - antiviral resistance

KW - boceprevir

KW - COVID-19

KW - Mpro

KW - Mpro inhibitor

KW - nirmatrelvir

KW - protease inhibitor

KW - SARS-CoV-2

U2 - 10.3390/v15091970

DO - 10.3390/v15091970

M3 - Journal article

C2 - 37766376

AN - SCOPUS:85172447987

VL - 15

JO - Viruses

JF - Viruses

SN - 1999-4915

IS - 9

M1 - 1970

ER -