The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

Fig. 1. Inactivation of the Leader protein reduces EV-enclosed virus release by EMCV-infected cells. a, b EMCV-Wt and EMCV-LZn-infected HeLa cells and corresponding cell culture supernatants were harvested at the indicated timepoints after infection. Line graphs display the increase in intracellular virus titers (a) and virus titers in the cell culture supernatant (b) over time, as determined by end-point dilution. Depicted are mean values ± SD from n = 3 independent experiments. c 10 K and 100 K EVs were separated from naked virions using density gradient centrifugation and EV-enclosed virus titers were determined by end-point dilution assay. Bars display the mean fold infectivity of EMCV-LZn EVs relative to EMCV-Wt EVs ± SD from n = 5 independent experiments. ****p < 0.0001, as assessed by a two-tailed one-sample t-test. ND not detected. d 100 K EVs released by cells infected with either EMCV-Wt or EMCV-LZn were isolated and EV-protein composition was analyzed by LC-MS/MS. The number of unique EMCV peptides identified within and outside of the capsid-coding region are indicated. Source data are provided as a Source Data file.

Fig. 2. The EMCV Leader is required for the induction of an infectious EV subpopulation with an FSC-high phenotype. a 10,000×g (10 K) and 100,000×g (100 K) ultracentrifugation pellets were collected from an equal number of EV-producing cells 8 h after mock, EMCV-Wt, or EMCV-LZn infection and analyzed by western blotting for the presence of EV-marker proteins CD9, CD63, and flotillin. Depicted is a representative of n = 3 independent experiments. b–g EVs were isolated from cell culture supernatants of mock, EMCV-Wt, and EMCV-LZn infected cells by differential ultracentrifugation and density gradient centrifugation at indicated timepoints p.i. EVs were fluorescently labeled with CFSE and analyzed by high-resolution flow cytometry. b The contribution of 10 K vs. 100 K EVs to the overall EV release by mock, EMCV-Wt, and EMCV-LZn infected cells was quantified. Mean values ± SD are presented for n = 3 independent experiments. **left p = 0.0025, **right p = 0.0015. c, d Depicted is the increase over time of 100 K EV numbers present in 1.06–1.10 g/ml fractions measured in a fixed time window of 30 s (c) and the number of EVs present in those density fractions at 8 h p.i. (d). ** p = 0.0047, *p =…

Fig. 3. Analysis of the EV proteome supports a Leader-dependent role for secretory autophagy in the formation of virus-induced EVs. EVs released by cells infected with either EMCV-Wt or EMCV-LZn were isolated and EV-protein composition was analyzed by LC-MS/MS. a Venn diagram depicting the number of unique and overlapping host proteins identified in at least 2 out of 3 replicate preparations of EMCV-Wt and EMCV-LZn EVs. b–e Functional enrichment analysis for the 44 unique proteins in EMCV-Wt EVs was combined with an evaluation of protein–protein interactions using the STRING database. b, d Functional annotations are listed for the GO domains cellular component (b) and biological process (d). Bars depict the fold enrichment for the indicated GO-terms compared to the genome, with concomitant p values as assessed by one-tailed Fisher’s exact test with Bonferroni correction for multiple testing. c, e Proteins in interactome networks are indicated by their gene name and thickness of the connecting lines correlates with the strength of data support for interaction. Color-coding of individual proteins corresponds to their association with GO-terms that are enriched >10-fold compared to the…

Fig. 4. The EMCV Leader promotes the accumulation of autophagosomes during infection. a Mock-infected cells and cells infected with EMCV-Wt or EMCV-LZn were harvested 8 h p.i. Whole-cell lysates were analyzed for conversion of LC3I to LC3II by western blotting and the LC3II:LC3I ratios were calculated relative to mock-infected cells. *Indicates a detected LC3 band with intermediate size. Actin was included as a loading control and EMCV-3D to confirm infection. Depicted are representative blots and the mean ± SD of n = 4 independent experiments. *p = 0.0341 (bottom), p = 0.0257 (top) as determined by a two-tailed one-sample t-test. # p = 0.0437 using a two-tailed t-test. b Schematic overview of the mCherry-EGFP-LC3 autophagy reporter system. Using spot detection on LC3 reporter cells imaged by confocal microscopy, the accumulation of LC3 in autophagosomal vs. autolysosomal compartments can be monitored. Whereas autophagosomes appear as mCherry+ EGFP+ LC3+ puncta, autolysosomes are detected as mCherry+ EGFP− LC3+ puncta due to a loss of EGFP fluorescence in the acidic environment of the autolysosome lumen. c LC3 reporter cells were infected with EMCV-Wt or EMCV-LZn for the indicated time…

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…

Fig. 6. Apilimod reactivates secretory autophagy and promotes EV-enclosed virus release in the absence of the EMCV Leader. a EMCV-LZn infected cells were treated with 500 nM apilimod for 16 h prior to infection or 200 nM BafA1 1 h p.i. onwards. EV-containing 100,000×g ultracentrifugation pellets were isolated from supernatants of an equal number of cells at 8 h p.i. and analyzed by western blot for the presence of extracellular released LC3. Depicted is a representative image of n = 2 independent experiments. LC3I PC and LC3II PC are positive control samples for western blot detection of LC3I and LC3II, respectively. b 100 K EVs were labeled with CFSE, purified by density gradient centrifugation, and analyzed by high-resolution flow cytometry. Bars indicate the mean fold increase in EV release relative to untreated controls ±SD from n = 3 (BafA1) or n = 4 (apilimod) independent experiments. **p = 0.0064, ns (left) p = 0.1148 as assessed by a two-tailed one-sample t-test, or two-tailed t-test for ns (right) p = 0.9936. c, d Depicted is the mean fold increase in EV-enclosed infectivity (c) and intracellular virus titers (d) relative to untreated controls ±SD corresponding to the samples in…