Fig. 4: Mutations near glycosylation sites affect the suppression of furin cleavage by GalNAc-T3 and T7.

a The sequence alignment of spike protein furin site from SARS-CoV-2 variants. b MALDI-TOF analysis of GalNAcylation reactions catalyzed by GalNAc-T3 and T7 on the synthetic multibasic substrate of SARS-CoV-2 spike protein (674-693) with P681H and P681H/N679K mutations. Reactions were performed and analyzed as in Fig. 1. c Western blot and quantitative analysis of the P681H and P681H/N679K spike protein processing in HEK293T WT and GALNTs KI cells. Plasmids encoding native, P681H or P681H/ N679K spike protein were transfected into HEK293T WT and GALNT3/T7 KI cells as indicated on top of the blot and spike proteins were detected with anti-S2 antibody. The results here are representative blots from three independent experiments. Quantitative analysis of Spike processing was performed by measuring the densitometry ratio between Cleaved-S and all forms of full-length S using Image J. Data are presented as mean values ± SD (n = 3 independent experiments). d Syncytium formation results of the P681H and P681H/N679K spike protein in Vero E6 cells. Vero E6 cells transfected with vector (NC), S-P681H, S-P681H/GALNT3/T7, S-P681H/N679K or, S-P681H/N679K/GALNT3/T7 plasmids were stained with DAPI (blue) and immuno-stained with anti-Flag antibody (green). The wild-type spike (S-Native) was included as a positive control. Syncytia formation was quantified by the number of nuclei per syncytium (FITC⁺ cells containing multiple nuclei). The N values indicate the number of syncytium counted. Data are presented as mean ± SD. n = 4 independent experiments. For statistical comparisons between means in data (c, d), two-tailed P values are calculated by unpaired Student’s t test. Unless otherwise labeled, the displayed P values are the significance between the experimental group and the control group (HEK293T or S-Native). NS: not significant. e Western blot analysis of the assembly of SARS-CoV-2 VLPs with P681H or P681H/N679K spike protein in HEK293T WT and GALNT3/T7 KI cells. VLPs with the native sequence of spike protein were produced in parallel as a control. The results here are representative blots from three independent experiments. Source data are provided as a Source Data file.