Fig 4. Reduced infectivity of SARS-CoV-2 pseudoviruses expressing mutated spikes.

(A) Schematic representation of the ten spike variants that were tested for infectivity. Amino acids substitution in each spike mutants is shown in the figure. NTD = N-terminal domain, RBD = receptor binding domain, FP = fusion peptide, HR1 = heptad repeat 1, HR2 = heptad repeat 2, TM = transmembrane domain, CT = cytoplasmic domain. (B) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The y-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the x-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. (C) FACS staining of 293T-ACE2 cells. The gray histogram shows the staining of the 293T-ACE2 cells with secondary antibody only. The empty black histograms depict the staining anti-ACE2 antibody or RBD-Ig. Shown is one representative experiment out of three preformed. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated spikes, unmutated (Wuhan-Hu-1) spike protein and bald pseudovirus. The x-axis depicts the mutation in the spike of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, *p < 0.05, **p < 0.005). Figure was generated using biorender.com.