Figure 3.
VSV-SARS-CoV-2 Spike Particles Activate and Use the Acid Sphingomyelinase for Infection
(A) Genetic downregulation of acid sphingomyelinase (ASM) with short hairpin RNA (shRNA) prevented infection of Caco-2 cells with pp-VSV-SARS-CoV-2 spike but had no impact on infection with particles that express the pp-VSV-G. Downregulation of neutral sphingomyelinase 2 (NSM2) did not affect the infection of Caco-2 cells with pp-VSV-SARS-CoV-2 spike. Control shRNA did not change the infection of Caco-2 cells by pp-VSV-SARS-CoV-2 spike. Cells were infected with pp-VSV-SARS-CoV-2 spike or pp-VSV-G, and cells positive for EGFP were counted in 2,000 cells per sample. Downregulation of ASM or NSM was confirmed by measuring the activity of the enzymes (right panels).
(B) Vero cells were infected with pp-VSV-SARS-CoV-2 spike, pp-VSV, or pp-VSV-G for the indicated times or were left uninfected. Samples were pre-incubated with AT or with the solvent (0.9% NaCl) or were left untreated. In addition, recombinant (rec.) ACE2 protein or neutralizing anti-spike antibodies was added prior to and maintained during the infection. Cells were lysed in 250 mM sodium acetate (pH 5.0) and 0.2% NP40. ASM activity was determined by measuring the consumption of [14C]sphingomyelin.
Shown are the means ± SD of the percentage of infected cells or the activity of the ASM or NSM from 6 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests.