Fig 3. Removal of the glycosylated MUC1 extracellular domain enhances SARS-CoV-2 entry.

(A) Microscopy images of Calu-3 cells treated with StcE, E447D, neuraminidase or fucosidase infected with SARS2-S pseudotyped VSV-GFP without or with neutralizing monoclonal antibody (mAb) against SARS2-Spike. White scale bars represent 200 μm. (B) Quantification of SARS2-S pseudotyped VSV-GFP signal in Calu-3 cells using EVOS software. StcE treatment resulted in a 5.4-fold increase in infection. (C) Quantification of luciferase signal (RLU) in Calu-3 cells after treatment with indicated enzymes and infection with SARS2-S pseudotyped VSV-Luc in the absence or presence of mAb against spike. A 4-fold increase in RLU value was observed when cells were treated with StcE. (D) Quantification of Calu-3 cell infection with VSV-G pseudotyped VSV-Luc lacking the spike protein. Infection was not blocked by the anti- spike mAb. (E) Infection of Calu-3 cells with authentic SARS-CoV-2 after treatment with indicated enzymes. StcE treatment resulted in a 2-fold increase in infected cell count. Neuraminidase and fucosidase treatment did not significantly impact viral entry. Represented values are the mean ± SEM of three biological replicates performed in triplicate. Statistical analysis was performed by repeated measures one way-ANOVA with Dunnett’s post-hoc test. p > 0.05 [ns, not significant], p<0.05 [*], p<0.01 [**], p<0.001 [***], p<0.0001 [****].