Fig. 2: VSVΔG-GFP pseudotyped virus assay.

A Generation of VSVΔG-GFP pseudotyped particles. HEK293T cells were transfected with the corresponding SARS-CoV-2 S protein mutant and infected with a VSV lacking the G gene and expressing GFP (VSVΔG-GFP). The resulting virus, incorporating the S mutants on its surface (VSVΔG-GFP -S), was recovered from the media and used to infect Vero-TMPRSS2 cells. Viral infectivity was then analyzed by counting GFP-positive cells. B–D Focus forming units (FFU) measured after infection with VSVΔG-GFP pseudotyped with different mutants of the SARS-CoV-2 S protein TMD. All samples were normalized to the wild-type SARS-CoV-2 S protein (WT). The p-values (<0.05) for individual one-sample t-tests (vs. WT) are indicated above each bar. B FFU from VSVΔG-GFP particles decorated with the wild-type SARS-CoV-2 S protein (WT), the SARS-CoV-2 S protein where TMD was eliminated (ΔTMD), and a scrambled version of the SARS-CoV-2 S protein TMD (SCRBL). C FFU from pseudotyped VSVΔG-GFP with the SARS-CoV-2 S protein with substitutions of amino acid stretches, Phe₁₂₂₀ to Gly₁₂₂₃, Leu₁₂₂₄ to Ile₁₂₂₇, and Val₁₂₂₈ to Thr₁₂₃₁, with alanine (Phe₁₂₂₀-Gly₁₂₂₃A, Leu₁₂₂₄-Ile₁₂₂₇A, and Val_1228-Thr₁₂₃₁A, respectively). Substitutions of amino acids Phe₁₂₂₀ to Gly₁₂₂₃ to leucine (Phe₁₂₂₀ to Gly₁₂₂₃L) were also included. D FFU from pseudotyped VSVΔG-GFP with the SARS-CoV-2 S protein, including alanine insertions at positions 1221, 1226, 1228, 1230, and 1232 (InsA₁₂₂₁, InsA₁₂₂₆, InsA₁₂₂₈, InsA₁₂₃₀, and InsA₁₂₃₂), tryptophan insertion at position 1221 (InsW₁₂₂₁), and methionine insertion at position 1230 (InsM₁₂₃₀). Four independent biological replicates were performed.