SARS-CoV-2 Spike Protein Fusion Assay

Cell–cell fusion assays were conducted as previously described [9 (link), 29 (link)]. Briefly, HEK293T cells transiently transfected with hACE2 and TMPRSS2 were used as target cells, and HEK293T cells transiently transfected with SARS-CoV-2 S protein and GFP were used as effector cells. 40 h after transfection, target cells were treated with vehicle, TAT or peptide (10 µM) 15 min before adding the effector cells. The effector cells were detached from their culture dishes with 0.25% trypsin and overlaid onto a target cell monolayer at an effector:target cells ratio of 1:3. After 2 h incubation, 10–21 images were randomly taken to count the number of fused vs. unfused cells using fluorescence microscopy (Bio-Rad). The cells number were counted using ImageJ (NIH). Effector cells incubated in DMEM supplemented with 10% FBS without target cells were used as a negative control. All experiments were performed in triplicate. This assay was performed in a double-blind fashion.