Monitoring fusion kinetics of viral and target cell membranes in living cells using a SARS-CoV-2 spike-protein-mediated membrane fusion assay

Figure 1. SARS-CoV-2 S-protein-mediated membrane fusion assay Schematic showing that luminescence (RL) and fluorescence (GFP) only occur when the fragments of the split RL and GFP protein are reconstituted in the same cytosol after fusion.

Figure 2. Maps of expression vectors used in this study (A) The map of a vector expressing SARS-CoV-2 B.1.1 S protein harboring a D614G mutation, pC-SARS-2 S D614G. The SARS-CoV-2 Delta and Omicron BA.1 S protein expression vector maps are the same except for the size of each S gene. The B.1.1, Delta, and Omicron BA.1 S genes are 3822 bp, 3816 bp, and 3813 bp, respectively. (B) The pC-ACE2 vector map. (C) The pC-TMPRSS2 vector map. (D) The pDSP1–7 vector map. (E) The pDSP8–11 vector map. All vector maps were created by SnapGene.

Figure 3. Surface expression levels of S protein (A) Representative FACS data of surface S proteins in HEK293 cells. S protein-expressing-HEK293 cells were harvested at 48 h posttransfection and stained using rabbit anti-SARS-CoV-2 S S1/S2 polyclonal antibody. Expression levels of the indicated S proteins on the cell surface were measured by flow cytometry and are shown as the MFI in the histogram. Normal rabbit IgG was used as isotype control (gray). pCAGGS was used as the vector. The parent S protein was derived from the B.1.1 variant bearing a D614G mutation. (B) A summary of S protein expression levels on the HEK293 cell surface. The graph indicates the mean ± standard deviation (SD) MFI (n = 3). pCAGGS was used as a vector. The parent S protein was derived from the B.1.1 variant bearing a D614G mutation. Statistical significance was assessed using two-sided paired t test. ∗ p < 0.05 compared with the parent S protein (blue) and Omicron BA.1 S protein (green).

Figure 4. Representative S-protein-mediated membrane fusion assay in various cell lines S protein-mediated membrane fusion assay was performed in HEK293-ACE2, HEK293-ACE2/TMPRSS2, Calu-3/DSP1-7, HeLa-ACE2/TMPRSS2, HOS-ACE2/TMPRSS2 and VeroE6/TMPRSS2 cells, respectively. Luminescence, indicating successful cell–cell fusion, was monitored at 0, 6, 12, 18, and 24 h after mixing the effector and target cells. The experiments were performed in quadruplicate. The parent S protein was derived from the B.1.1 variant bearing a D614G mutation. Fusion activity (arbitrary unit) was measured as luminescence normalized to MFI and is shown as mean ± SD. Statistical differences between a pair of variants across timepoints were determined by multiple regression and indicated in each graph. The 0 h data were excluded from the analyses. The FWERs calculated using the Holm method are indicated in the figures.

Figure 5. Cleavage of S protein in HEK293 cells (A) Representative immunoblots. The cell lysates of HEK293 cells transfected with the S gene were subjected to immunoblotting. The indicated S proteins were detected by mouse anti-SARS-CoV-2 S monoclonal antibody (S2 subunit). pCAGGS was used as the vector. The parent S protein was derived from the B.1.1 variant bearing a D614G mutation. β-actin was used as the loading control. (B) Summary of the S1/S2 cleavage efficiency of the indicated S proteins in HEK293 cells. Full-length S and S2 band intensities were quantified and the ratio of S2 to the sum of S and S2 [S2/(S + S2)] was calculated from three independent experiments. The parent S protein was derived from the B.1.1 variant bearing a D614G mutation. Data represent the mean S2/(S + S2) ± SD relative to the parent S protein. Statistical significance was performed by a two-sided paired t test. ∗ p < 0.05 compared with the parent S protein (blue), Delta S protein (red), and Omicron BA.1 S protein (green).