Fig. 4: PS-rich EVs from body fluids interfere with viral apoptotic mimicry.
a, Inhibition of ZIKV infection by titrated EVs and control PC-liposomes. n = 3 biological replicates, mean values ± standard deviation (s.d.). See Extended Data Fig. 4a for cytotoxicity. Right: IC50 calculated by non-linear regression. Each data point indicates a separate EV isolation (n = 4 (blood), n = 3 (semen, saliva), n = 1 (urine, milk and liposomes)) analysed in biological triplicates. One-way ANOVA with Bonferroni’s post-test, 95% confidence interval. b, Cell attachment of Zika virions (left) or hybrid vesicles (right) after addition of Cy5-fluorescent semen EV- or PC-liposome hybrids. See Extended Data Fig. 4c for labelling workflow and Extended Data Fig. 4d–f for characterization of hybrid vesicles. Quantification of three maximum-intensity projections from 25 slices per condition (n = 3), mean ± s.d. (for liposomes-only control, see Extended Data Fig. 4g,h). Unpaired, two-tailed t-test, 95% confidence interval. All without indication NS (P > 0.05). c, Confocal maximum intensity projections of Zika virions (green) and hybrid vesicles (red) on cells (blue nuclei) as used for b, representative of three images; scale bars, 20 μm. d, Anti-ZIKV (MOI 0.25) activity of hybrid semen EVs and occupation of Axl determined by confocal microscopy via M2 overlap coefficients for EV-Cy5 and Axl-PE, means ± standard error of the mean (s.e.m.) (n = 3 z-stacks). Representative image of three shown in e. See Extended Data Fig. 5a for images of all concentrations; scale bar, 20 μm. f–i, EVs treated with phospholipase D (‘Shave’), LA (‘+Block’), followed by incubation (‘+Incubated’). PS reintroduced to EVs by cyclodextrin treatment (‘+Resupply’). f, Assessment of ‘Shaving’ (S) by iodine-stained HPTLC. g, Bead-assisted flow cytometry assessing the PS exposure on CD9, CD63 and CD81 bead-bound EVs by LA staining in comparison with CD9/CD63/CD81 (TSPN) staining. n = 5 (Untreated; Shave + Block + Resupply), n = 4 (Shave + Block) or n = 3 (Shave + Block + Incubated) separately treated EVs, mean values. h, Anti-ZIKV (MOI 0.25) activity, representative dataset of biological triplicates, means ± s.d., with corresponding IC50 values in i for n = 5 separately treated vesicle preparations, means ± standard error of the mean. One-way ANOVA with Bonferroni’s post test (95% CI).
