Fig. 4: Decreasing Sia on ACE2+ cells decreases RBD binding and SARS-CoV-2 pseudotyped lentiviral infection.
From: Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2
a,b, Changes in Sia levels from 3FNeu5Ac treatment of HEK293 ACE2+ cells determined by SNA (a) and PNA (b) lectin staining by flow cytometry. These n = 3 biological replicates are represented as mean fluorescence intensity (MFI) values with 95% confidence interval (95% CI) error. c, 3FNeu5Ac treatment decreases RBD binding. The MFI values for n = 3 biological replicates within a single experiment (left) and the average of n = 3 independent experiments (right) are shown. The average RBD binding to control cells was set to 100%. d, 3FNeu5Ac treatment decreases SARS-CoV-2 pseudovirus infection of HEK293 ACE2+ cells. The percentage of GFP+ cells for n = 3 biological replicates within a single experiment (left) and the averages of n = 3 independent experiments (right) are shown. The error bars associated with the single experiment are 95% CI. e, ACE2 expression level does not change following 3FNeu5Ac treatment. The MFI with 95% CI for n = 3 biological replicates is shown. f–j, Flow cytometry results for changes in Sia levels within CMAS–/– ACE2+ HEK293 cells determined by SNA (f) and PNA (g) lectin staining, RBD binding (h), SARS-CoV-2 pseudovirus infection (i) and ACE2 expression (j) on CMAS+ versus CMAS–/– ACE2+ HEK293 cells. Each point represents the average of n = 3 biological replicates for each individual clone with 95% CI error bars. k, SARS-CoV-2 pseudovirus infection of CMAS+ versus CMAS–/– ACE2+ HEK293 cells treated with or without 3FNeu5Ac. The percentage of GFP+ cells for n = 3 biological replicates with 95% CI error bars is shown. l,m, Pretreatment of Vero-E6 cells (l) and ferret lung tissue sections (m) with neuraminidase decreases RBD binding. Scale bars represent 100 μm (l) and 200 μm (m), and results represent data from one representative sample from n = 5 experiments. n, Quantification of fluorescence intensity for RBD binding and lectin staining of Vero-E6 cells and ferret lung tissue sections. Error bars represent ±s.d. of n = 3 replicates. Statistical significance was calculated based on either two-tailed unpaired Student’s t-test (a–k) or analysis of variance (ANOVA) with multiple comparisons (l,m). Flow cytometry data were analyzed on FlowJo version 9.9.6, and graphs were plotted on GraphPad Prism 8; NS, not significant.
