Glycobuffer

Two milliliters of SARS-CoV-2 P4 supernatant containing 2 × 10⁷ PFU/mL was purified using an Amicon Ultra column (molecular size cutoff, 100 kDa; Merck) by centrifugation and washing in PBS at 2,000 × g; 40 mL purified supernatant was combined with 2,500 U recombinant glycerol-free PNGase F and Glycobuffer 2 (New England Biolabs) by following the manufacturer’s protocol for nondenaturing digestion and incubated at 37°C for 5 h. Parallel controls included incubation of purified virions with or without PNGase F and Glycobuffer, incubation with heat-inactivated (75°C for 10 min) PNGase F, and incubation with PNGase F and Glycobuffer during virus inoculation of host cells for 30 min. After incubation, samples were either directly used for infection or combined with radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors to generate lysates for blotting. Alternatively, Vero E6 cells were preincubated with or without 100,000 U/mL PNGase F in serum-free Opti-MEM for 5 h at 37°C and 5% CO₂ before being either inoculated with SARS-CoV-2 or surface biotinylated. To biotinylate surface proteins, cells were washed in PBS, pH 8, incubated with 2.5 mM EZ-link NHS-biotin (ThermoFisher) for 30 min on ice, washed in PBS–100 mM glycine, and lysed in RIPA buffer and protease inhibitors as described above.

PNGase F and O-Glycosidase was purchased from NEB. Briefly, 2 μg of purified S1 protein was added with 1 μL of 10X glycoprotein denaturing buffer (NEB) in total of 10 μL of reaction volume and denatured at 95 °C for 5 min. Then, the mixture was chilled on ice for 30 s followed by centrifugation for 10 s at 10,000 X g. Reaction volume was increased to 20 μL by adding 2 μL 10X GlycoBuffer (NEB), 2 μL 10 % NP40, water and 1 μL of enzyme PNGase F or 2 μL of O-Glycosidase and incubated at 37 °C for 1 h. The extent of glycosylation was analyzed by mobility shift on SDS-PAGE followed by Western blotting.

Nine microliter of MVB072-labelled tomato root extract and Bovine Fetuin (Promega) were treated with 1 μl of 10X glycoprotein denaturing buffer (New England BioLabs) and heated at 95°C for 5 min. The denatured proteins were chilled on ice. Two microliter 10X GlycoBuffer (New England BioLabs), 2 μl 10% NP40 (Promega), and 6 μl H₂O was added to the reaction. The mixture was treated with 1 μl PNGase F (New England BioLabs) or with 1 μl H₂O and incubated at 37°C for 1 h. Samples were analyzed on 16% SDS-PAGE.