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. 2020 Oct 26;9:e61552. doi: 10.7554/eLife.61552

Figure 4. N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells.

Figure 4.

(A) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O]- and [N]-293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. (B–C) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. (D) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. (E) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N]-293T products due to truncation of glycan biosynthesis. (F) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N]-293Ts is almost fully proteolyzed during viral production (red arrowhead). *p<0.05 with respect to all other treatments.