Figure 5. Mannosidase-I blockade using kifunensine inhibits SARS-CoV-2 pseudovirus entry into 293T/ACE2 cells.

(A) VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15 µM kifunensine or vehicle control. The six viruses were added to 293T/ACE2 at equal titer. (B–D) Microscopy (panel B) and cytometry (panel C, D) show ~90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (*p<0.05). (E) Spike molecular mass is reduced in the western blots due to high-mannose glycan synthesis in runs with kifunensine. Intact Spike is reduced in the presence of kifunensine, in anti-FLAG blot. (F) The polybasic furin ‘RRAR’ site was substituted by a single ‘A’ amino acid in Spike-delta. Virus with Spike-delta were expressed both in the presence of vehicle and kifunensine. Western blot shows lack of S1-S2 cleavage in this construct. In viral entry assay, kifunensine reduced DsRed expression in 293T/ACE2 cells, even in the case of Spike-delta pseudovirus (*p<0.05). Similar observation was made at two different viral titers (0.3 and 0.6 μg/mL p24 equivalent).

Figure 5—figure supplement 1. Effect of kifunensine.

(A) Lectin binding upon kifunensine treatment. HEK293T cells were cultured with varying concentrations of kifunensine or vehicle for 2 days. PHA-L binding measurement shows marked reduction in the cell-surface expression of complex glycans and also a decrease in ECL engagement (recognizes lactosamine chains). Kifunensine did not affect cell growth rate or viability. It also did not reduce pseudovirus production titers. Data are Mean ± STD (N = 3). (B) Kifunensine reduced Spike viral infection. Bright field images for cell monolayer (left) for microscopy data shown in main manuscript (reproduced in right panels). Kifunensine reduces Spike-WT and Spike-mutant pseudoviral entry by ~85–90%.