Fig. 5: Analysis of SARS-CoV-2 S protein membrane fusion properties by BiMuC.
A Schematic representation of the BiMuC assay. The Venus fluorescence protein (VFP) can be split into two fragments, VN and VC, respectively, neither of which is fluorescent. These two fragments were fused to cJun and bFos (VN-cJun and VC-bFos) and transfected into HEK 293 T cells in separate cell pools. Cells were co-transfected with the viral machinery required for membrane fusion, the corresponding SARS-CoV-2 S protein, and the ACE2 receptor. Additionally, both cell pools were transfected with the Renilla Luciferase (luciferase) to normalize the fluorescent values. Only functional SARS-CoV-2 S proteins facilitated the S-ACE2 recognition and membrane fusion, allowing VFP reconstitution and fluorescence. B Representative images of the assay where cells have been transfected with the SARS-CoV-2 S protein (WT) or the SARS-CoV-2 S protein bearing a scrambled version of the TMD (SCRBL). Scale bar = 100 microns. C–E The fluorescence/luciferase signal ratio (GFP/Luc ratio) was measured for the SARS-CoV-2 S protein mutants tested on this assay. Samples were normalized to the SARS-CoV-2 S protein WT. Significative p-values (<0.05) for individual one-sample t-tests are indicated above each bar. C GFP/Luc ratio for the SARS-CoV-2 S protein (WT), the SARS-CoV-2 S protein where TMD was eliminated (ΔTMD), and a chimera bearing a scrambled version of the SARS-CoV-2 S protein TMD (SCRBL). As a negative control, we also included cells that did not incorporate the SARS-CoV-2 S protein (ΔS). D GFP/Luc ratio for the SARS-CoV-2 S protein with substitutions of 4 amino acid stretches Phe1220 to Gly1223, and Val1228 to Thr1231, by alanines (Phe1220-Gly1223A and Val1228-Thr1231A), and the SARS-CoV-2 S protein including alanine insertions in positions 1221, 1228, and 1232 (InsA1221, InsA1228, and InsA1232). E GFP/Luc ratio for the SARS-CoV-2 S protein bearing the following point mutations: Gly 1219 to Ile and Gly1223 to Ile, Ile1221 to Tyr, Ile1225 to Tyr, Met1229 to Tyr, Ala1222 to Tyr and Gly1223 to Tyr, and Leu1224 to Tyr and Val1228 to Tyr (G1219I G1223I, I1221Y, I1225Y, M1229Y, A1222Y G1223Y, and L1224Y V1228Y, respectively). All panels include the WT values used to normalize the data (dotted line) and the SCRBL samples. F Correlation of FFU measured in the VSVΔG-GFP infection assay against values of reconstituted GFP/Luc ratio in the BiMuC fusion assay. Colors show samples that have lower signals than WT values in both systems (pink background), higher in both systems (green background), or different between the two assays (white background). At least three independent biological replicates were performed.
