Synchronized Viral Entry Kinetics

Synchronized infection experiments were performed to determine entry efficiency of the virus variants. Infection of cells was performed on ice and cells were immediately transferred to 4°C for 1 hour after virus dilutions were added to ensure synchronized virus uptake and start of replication. After virus adsorption, cells were washed 5 times with PBS to remove excess virus particles. Cells were lysed either immediately or incubated with infection medium until 4 or 6 hours postinfection. At the indicated time points, medium was removed and cells were lysed with MagNA Pure 96 external lysis buffer (Roche, Penzberg, Germany). Isolation of RNA from cell lysates and quantitative RT-PCR on subgenomic nucleocapsid RNA was performed as described elsewhere [69 (link),70 (link)]. Entry inhibitors were dissolved in DMSO in the indicated concentrations, added 1 hour prior to virus infection, and were supplied for the entire duration of the experiment. Pretreatment of virions with TPCK-treated trypsin (Sigma Aldrich) or saliva (1:10 diluted in serum-free medium) was performed for 1 hour at 37°C prior to virus infection.