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. 2021 Nov 25;74:103712. doi: 10.1016/j.ebiom.2021.103712

Figure 2.

Figure 2

N-glycosylation in spike is critical to viral infection. (a) Diagram of the spike pseudovirus package. (b) Effect of the deglycosylated spike on viral infection. Luciferase activity was measured to evaluate pseudotype viral entry ability in HEK293T-ACE2 cells challenged with SARS-CoV-2, Alpha, or Beta variant spike pseudovirus for 2 days. Before infection, pseudovirus were pretreated with PNGase F, or Endo H by a non-denaturing reaction protocol (https://international.neb.com/protocols/2014/07/31/pngase-f-protocol). The control represents pseudovirus treated with glycosidase-free glycobuffer. Glycosylated and de-glycosylated S protein molecular weight were assessed by Western blot. n=3. Statistical method: two-way ANOVA, Tukey post hoc tests, ***p < 0.001. (c) Diagram of the NXT motifs in spike S1 of CoV-2, CoV-2 4NQ, CoV, and CoV 3N (left panel). Luciferase activity was measured in HEK293T-ACE2 cells infected with distinct pseudovirus generated from wild-type and mutant spike SARS-CoV or SARS-CoV-2 (right panel). Western blotting was performed to examine S protein molecular weight in CoV-2, CoV-2 4NQ, CoV, and CoV 3N. ***p < 0.001 (One-way ANOVA, Tukey post hoc tests). (d) The infectivity of pseudoviruses generated from DMSO or NGI-1-treated HEK293T cells was evaluated by luciferase assay (red). Cell viability was determined by MTT assay (black bar). n = 3 with mean ± SD shown. ***p < 0.001 (One-way ANOVA, Tukey post hoc tests).