Fig. 5: Enhancing autophagic degradation does not inhibit Leader-induced secretory autophagy and EV-enclosed virus release.

EMCV-Wt infected cells were treated with 200 nM rapamycin 1 h p.i. onwards to increase autophagic flux and enhance autolysosome formation. a Representative confocal microscopy images of infected mCherry-EGFP-LC3 reporter cells 6 h p.i. Bar graphs display the ratio between the total surface area of EGFP and mCherry positive puncta, that correspond to autophagosomes (EGFP+ mCherry+) and autolysosomes (EGFP−mCherry+). Indicated are mean values ± SEM of ≥7 separate images with 2–5 cells each from a representative of n = 3 individual experiments. ***p = 0.0008, *p = 0.0434 as assessed by one-way ANOVA with Tukey’s multiple comparison test. Scale bar = 10 μm. b Western blot analysis of LC3 release in ultracentrifugation pellets harvested 8 h p.i. from cell supernatants of an equal number of cells. Depicted is a representative image of n = 3 independent experiments. LC3I PC and LC3II PC are positive control samples for western blot detection of LC3I and LC3II, respectively. c, d 10 K and 100 K EVs were isolated and purified using density gradients. Depicted is the total amount of EV-associated infectivity (c) and its distribution over 100 K vs. 10 K EVs (d) (mean ± SD of n = 3 independent experiments) as determined by end-point dilution assay. ***p = 0.0009, ns p = 0.6599 as assessed by an unpaired, two-tailed t-test. Source data are provided as a Source Data file.