Figure 4.
VSV-SARS-CoV-2 Spike Particles Induce Ceramide Formation, Facilitating Infection
(A) To determine ceramide levels, we organically extracted the samples and quantified ceramide levels by using the ceramide kinase method. We quantified C16 (C16-Cer)/C18 ceramide and C22/C24 ceramide. If indicated, cells were preincubated with 5, 10, 20, or 25 μM AT for 4 h before the addition of pp-VSV-SARS-CoV-2 spike. The rec. ACE2 protein or neutralizing anti-spike antibodies were added as above. In addition, cells were infected with pp-VSV or pp-VSV-G.
Displayed are the means ± SD of the ceramide concentrations from each 5 independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests, as indicated or compared to the corresponding value without inhibitor.
(B) Flow cytometry reveals the formation of ceramide in the outer leaflet of the cell membrane. Cells were treated with 25 μM AT, each 2 μg rec. ACE2 protein or neutralizing anti-spike antibodies and infected with pp-VSV-SARS-CoV-2 spike. Controls were infected with pp-VSV or pp-VSV-G. Shown are the mean fluorescence values (in a.u.) ± SD of 6 independent flow cytometry studies; ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests.
(C) Reconstitution of ceramide in Vero cells (left panel) that had been treated with 10 or 25 μM AT or in Caco-2 cells (right panel) transfected with shRNA _targeting ASM by the addition of C16-Cer (10 μM) or ASM (0.2 U/mL) during the infection with pp-VSV-SARS-CoV-2 spike restores viral infection of the cells. The rec. ACE2 protein or neutralizing anti-spike antibodies prevented the infection. Displayed are the means ± SD of the percentage of infected cells from 6 independent experiments. ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests. See also Figure S3.