Figure 1. S2 stem N-glycans are critical for spike cell surface expression and ACE2-Fc functional binding.

A. Heat map of mutation prevalence across SARS-CoV-2 world health organization variants of concern and interest. Red asterisk highlights glycosylation sites lost in individual viral strains and blue asterisk highlights the new glycosylation sites that have appeared. Data are rendered using dashboard at outbreak.info, using GISAID data. Only mutations with >75% prevalence in a single lineage are plotted. Each lineage is sequenced at least 1000 times. B. Post-fusion S2 conformation of spike protein (PDB: 7E9T) with five S2 stem N-glycans distributed along the axis. The distance between adjacent glycans is indicated. Asn (N) to Gln (Q) Spike N-glycan mutants at N1098Q, N1134Q, N1158Q, N1173Q and N1194Q are designated G1, G2, G3, G4 and G5, respectively. All possible N-to-Q mutant combinations were produced as shown in the table. C. 31 spike mutants and parent spike were transiently expressed in 293T cells. Anti-S1 mAb and ACE2-Fc fusion protein binding were simultaneously detected on cell surface. Mutations at N1098 (G1, shown using green bars) reduced spike function, particularly in synergy with additional N-to-Q mutations at other sites. ACE2-Fc binding was abolished in G1234, G1345 and G12345 mutants. The relationship between cell surface spike expression and ACE2-Fc binding was linear (R² = 0.87). Selected G1-mutants, labeled red, were further analyzed in later studies. D. Western blots using anti-Flag and anti-S2 confirmed that stem N-glycan mutations reduce cell-surface expression, particularly upon implementing multiple edits. Anti β-Actin served as loading control. Note that some non-specific or Spike fragment bands appear when using cell lysates, but these are typically absent in virus blots. Densitometry was performed to quantify anti-Flag band intensity reduction. E. Anti-S1 and anti-S2 mAbs were applied in flow cytometry studies. The ratio of anti-S1 to anti-S2 mAb binding decreased in many cases suggesting enhanced S1 shedding upon implementing stem mutations. Data are Mean ± STD. * P<0.05, ** P<0.01 ,***P<0.001 with respect to parent.