A549 hace2 cells

The human A549-hACE2 cells, stably transfected to express the human ACE2 gene, were obtained from InvivoGen (San Diego, CA, USA) and cultured in Dulbecco’s modified eagle medium (DMEM) with 4.5 g/L glucose, 10% of heat-inactivated FBS (Fetal Bovine Serum), 2 mM L-glutamine, 100 U/mL Pen-Strep, and 10 mg/mL of Puromycin as selection antibiotic. Human CaCO2 cells were purchased from ATCC (American Type Culture Collection) and cultured in Eagle’s Minimal Essential Medium (EMEM) with 20% FBS, 2 mM L-glutamine, 100 U/mL Pen-Strep, and 1% non-essential amino acids. Normal Human lung fibroblast cells MRC5 were purchased from ATCC and cultured in DMEM with 4.5 g/L glucose, 10% of heat-inactivated FBS (Fetal Bovine Serum), 2 mM L-glutamine, and 100 U/mL Pen-Strep. All the cell lines were routinely grown in monolayers and maintained at 37 °C in 5% CO₂-humified atmosphere and regularly tested for mycoplasma presence.

Boceprevir and nirmatrelvir were synthesized by Acme Bioscience (Palo Alto, CA, USA) and dissolved and stored as previously described [47 (link)].
All cell culture experiments were carried out in African green monkey kidney VeroE6 cells kindly provided by J. Dubuisson and human lung carcinoma A549-hACE2 cells (InvivoGen, Toulouse, France), which were maintained as previously described [36 (link),47 (link),53 (link)].
The virus stock of the so-called original SARS-CoV-2 was derived from the patient isolate SARS-CoV-2/human/Denmark/DK-AHH1/2020 (GenBank: MZ049597), representing an originally circulating variant with the D614G substitution in the spike protein, and was generated as described previously [54 (link)]. Stocks of the polyclonal boceprevir escape viruses were obtained as described in “Selection for SARS-CoV-2 Resistance to Boceprevir”. Stocks of the recombinant SARS-CoV-2 mutants were obtained as described in “Generation of Recombinant SARS-CoV-2 Mutants”. All infectious cell culture experiments were carried out under biosafety conditions in accordance with Danish regulations and with permission from the Danish authorities.

VeroE6 cells (gift from J. Dubuisson) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Paisley, UK) as described (15 (link), 26 (link), 27 (link)). A549-hACE2 cells (InvivoGen, Toulouse, France) were cultured in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (Gibco, Paisley, UK) as described (15 (link), 26 (link), 27 (link)). Both culture media were supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml; Gibco/Invitrogen Corporation, Carlsbad, CA, USA). A549-hACE2 culture medium was in addition supplemented with puromycin (0.5 μg/ml; InvivoGen, Toulouse, France). Cells were maintained at 37°C and 5% CO₂ and subcultured every 2 to 3 days.