Figure 2. Sialic acid has modest effect on Spike binding and viral entry.

(A) Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2]. N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3], RBD [v4] and soluble ACE2 [v5]. All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. (B) Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. (C) Flow cytometry data showing S1-Fc (1.7 µg/mL) and RBD-Fc (0.35 µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4 µg/mL) binding (bottom). (D) Desialylation of Spike-protein expressed on 293 T/S had minimal effect on ACE2-Fc (0.7 µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2 µg/mL) and S1-Fc (1.7 µg/mL) by 26–56% (paired experiments, *p<0.05). (E) Pseudovirus with DsRed-reporter were developed with three different envelope proteins. VSVG pseudotyped virus infected both HEK293T (black line) and stable 293T/ACE2 (red line) cells. Virus with Spike-WT and Spike-mutant entered 293T/ACE2 only. (F) Same titer of virus (0.3 µg/mL p24-equivalent) were treated with or without sialidase, prior to addition to stable 293T/ACE2 cells. Infection using Spike-mutant was higher compared to Spike-WT. Sialidase treatment of virus had no effect. (G) 293T/ACE2 cells were sialidase treated prior to addition of VSVG (0.3 µg/mL p24-equiv.), Spike-WT (1.5 µg/mL p24-equiv.) or Spike-mutant (0.2 µg/mL p24-equiv.) pseudovirus. Sialidase treatment did not affect viral entry. Abbreviations: Spike signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), receptor-binding motif (RBM), subdomain 1 (SD1), subdomain 2 (SD2), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2) transmembrane section (TM), cytoplasmic tail (CT), ACE2: Angiotensin-converting enzyme-2; VSVG: Vesicular stomatitis virus G-protein; WT: wild-type; mut: mutant.

Figure 2—figure supplement 1. Sialidase treatment studies.

(A) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. (B) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. (C) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in Figure 2F (main manuscript). Viral entry was sialidase independent. (D) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in Figure 2G (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

Figure 2—figure supplement 2. p24 assay to determine viral titer.

(A) Representative calibration curve for p24 standard. (B) Absorbance value for viral stock in representative ELISA run. Nine different viruses were prepared and used in Figure 4 (main manuscript). All viruses were diluted 1:500,000 fold before ELISA measurement, except for VSVG virus made in [N]¯ 293 T cells (indicated by asterisk) which were diluted 1:50,000 fold, in this instance, due to lower titer.