Fig. 3: GalNAc-T3 and T7 inhibit furin-dependent assembly of the Virus Like Particles (VLPs). | Nature Communications

Fig. 3: GalNAc-T3 and T7 inhibit furin-dependent assembly of the Virus Like Particles (VLPs).

From: Sequential glycosylations at the multibasic cleavage site of SARS-CoV-2 spike protein regulate viral activity

Fig. 3

a Western blot and quantitative analysis of the assembly and release of SARS-CoV-2 VLPs in HEK293T WT and GALNTs KI cells. Different HEK293T cells were co-transfected with native S, together with HA-tagged M, E and N. VLPs and cell lysates were pelleted and analyzed separately. b Western blot and quantitative analysis of the assembly of SARS-CoV-2 VLPs with native S protein and mutants in HEK293T WT. The S protein mutant lacking furin site (S-R665A) failed to be incorporated into VLPs. c Western blot and quantitative analysis of the assembly of SARS-CoV-2 VLPs with native S protein and T678A/S686A mutant S protein in HEK293T WT and GALNTs KI cells. For (a, b, c), results here are representative blots from three independent experiments. Quantitative analysis of S protein incorporation into VLP was performed by calculating the densitometry ratio of S protein (cleaved-S plus FL-S) to N protein, and then normalizing it against the ratio calculated for HEK293T WT (a, c) or native S (b). Data are presented as mean values ± SD (n = 3 independent experiments). For statistical comparisons between means, 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. d The topology diagram of SARS-CoV-2 M protein. The N-terminal amino acid sequences of M proteins from SARS-CoV-2, SARS-CoV and MERS-CoV are aligned. The EE motif is highlighted by arrowheads. The diagram was generated by Protter76. e Western blot analysis of the assembly of SARS-CoV-2 VLPs with native M protein and its EE motif mutant in HEK293T WT. f Co-immunoprecipitation of S protein with native M protein and its EE motif mutant. Co-immunoprecipitation was performed using anti-HA mAb and examined by western blotting analyses using anti-S1 mAb. IgG were used as a control. For (e) and (f), the results here are representative blots from three independent experiments. Source data are provided as a Source Data file.

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