SARS-CoV-2 Pseudovirus Entry Assay

Pseudovirus production, infectivity, and entry assays were performed as described before (Mykytyn et al., 2021 (link)). Briefly, pseudoviruses expressing WT, MBCS mutant, and S686G S were titrated by preparing 10-fold serial dilutions in Opti-MEM I (1X) + GlutaMAX (Gibco). Thirty µl of each dilution was added to monolayers of 2 × 10⁴ VeroE6, VeroE6-TMPRSS2 or 8 × 10⁴ Calu-3 cells in the same medium in a 96-well plate. Titrations were performed in triplicate. Plates were incubated at 37°C overnight and then scanned on the Amersham Typhoon Biomolecular Imager (channel Cy2; resolution 10 µm; GE Healthcare). Entry routes were determined by pre-treating monolayers of VeroE6 or VeroE6-TMPRSS2 cells with a concentration range of camostat mesylate (Sigma) or E64D (MedChemExpress) diluted in Opti-MEM I (1X) + GlutaMAX (Gibco) for 2 hr prior to infection with 1 × 10³ pseudovirus. Plates were incubated at 37°C overnight and then scanned on the Amersham Typhoon Biomolecular Imager (channel Cy2; resolution 10 mm; GE Healthcare). All pseudovirus experiments were quantified using ImageQuant TL 8.2 image analysis software (GE Healthcare).