Fig 1.
The picornavirus security proteins CVB3 2Apro, TMEV L, and SAFV L promote EV-enclosed virus release with different efficiencies.
(A) Depicted is a schematic overview of the recombinant viruses used in this study. To generate these viruses, the coding sequences of TMEV L (LTMEV), SAFV L (LSAFV), or CVB3 2A (2Apro) proteins were fused to the polyprotein of an EMCV strain containing an inactivating mutation in the EMCV L protein (EMCV-LZn), separated by a 3C cleavage site. (B-G) HeLa cells were infected with the viruses described in (A) at MOI 10. (B) Intracellular virus titers were determined over time by end-point dilution assay and are depicted as line graph. In (C) a bar graph is depicted comparing the intracellular virus titers at 7 hpi. Corresponding extracellular virus titers are depicted in (D) and (C), with bar graphs comparing the extracellular virus titers at 7 hpi (left) and 10 hpi (right). (F) EVs released by cells infected with EMCV-Wt, EMCV-LZn, or EMCV-LZn reconstituted with the indicated viral proteins were isolated by density gradient centrifugation at 7 or 10 hpi. EV-enclosed virus titers were determined by end-point dilution assay. Data are expressed as fold increase in EV-enclosed virus titers relative to EMCV-LZn. (G) EV-enclosed virus release at 7 hpi was compared for EMCV-LZn reconstituted with proteolytically active vs. proteolytically inactive CVB3 2Apro (EMCV-2Apro and EMCV-2Amut respectively). Bar and line graphs depict means ± SD of n = 3–4 independent experiments. *p<0.05, ***p<0.0005, ****p<0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons test.
Fig 2.
CVB3 2Apro and EMCV L, but not TMEV L or SAFV L, strongly increase the release of virus-induced EV subsets carrying flotillin-1 and LC3.
EVs were isolated by means of density gradient centrifugation at 7 or 10 hpi with EMCV-Wt, EMCV-LZn, or the recombinant viruses EMCV-2Apro, EMCV-LTMEV, and EMCV-LSAFV. (A) EVs were fluorescently labeled with CFSE and quantified by high resolution flow cytometry. Depicted is the increase in EV numbers relative to the mock-infected control. Means ± SD of n = 3–4 independent experiments are shown. (B) Dot plots showing the forward (FSC) vs. side-scatter (SSC) profile of EVs in the peak fraction of samples corresponding to (A). (C) Bar graphs displaying the % of total EVs that fall within the FSChigh gate indicated in (B). (D) Western blot analysis of CD9, flotillin-1, and LC3 in density gradient purified EVs isolated from an equal number of cells. Depicted are representative images of n = 2 independent experiments. p**<0.005, ***p<0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons test.
Fig 3.
Suppression of PKR but not MAVS antiviral signaling potentiates the release of EVs and EV-enclosed virus during EMCV infection.
WT, MAVS KO, and PKR KO cells were infected with EMCV-Wt or EMCV-LZn. 7 hpi EVs were isolated by density gradient centrifugation to assess if inhibition of MAVS/PKR-mediated antiviral signaling cascades can rescue EV-enclosed virus release. (A) Intracellular and EV-enclosed virus titers were determined by end-point dilution assay. (B) Bar graphs displaying the fold difference in EV-enclosed virus titers detected in EMCV-LZn compared to EMCV-Wt infected samples, after correction for the corresponding differences in intracellular virus titers. Means ± SD of n≥3 independent experiments are shown. (C) EV numbers were quantified by high resolution flow cytometry. Depicted is the increase in EV numbers relative to the mock-infected controls. Means ± SD of n = 4 independent experiments are shown. (D) Dot plots showing the forward (FSC) vs. side-scatter (SSC) profile of EVs in the peak fraction of samples corresponding to (C). (E,F) EVs isolated by ultracentrifugation from an equal number of (mock) infected MAVS (E) or PKR KO cells (F) were analyzed for the presence of the common EV-associated proteins CD9 and flotillin-1. In addition, the extracellular release of LC3 was analyzed. Representative images of n≥2 independent experiments are shown. * p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001 based on a two-way one-sample t-test or two-sided t-test.
Fig 4.
The EMCV Leader requires the host kinase RSK to promote EV release during infection.
Hela-M WT cells, RSK-1/2/3 triple KO (TKO) cells, and TKO cells reconstituted with RSK-2 were mock infected or infected with EMCV-Wt. 7 hpi EVs were isolated from the supernatant of (mock) infected cells. (A) Intracellular virus titers and EV-enclosed virus titers were determined by end-point dilution assay. *** p<0.0005, * p<0.05 as determined by a RM one-way ANOVA with Geisser-Greenhouse correction. (B) Depicted is the fold change in EV-enclosed virus titers upon RSK TKO and RSK-2 reconstitution after correcting for the fold change in intracellular virus titers in the corresponding samples. * p<0.05 as determined by a two-tailed t-test or one-sample t-test. (C) EV numbers were quantified by high resolution flow cytometry. Depicted is the fold increase in EV numbers relative to the respective mock-infected controls. ** p<0.005 as determined by a two-tailed one-sample t-test. For (A-C) means ± SD of n = 5 independent experiments are shown.
Fig 5.
Activation of the stress kinase P38 MAPK in the presence of EMCV L and CVB3 2Apro, but not TMEV L and SAFV L, promotes EV and EV-enclosed virus release during infection.
(A) Phosphorylated P38 MAPK (Thr 180/Tyr 182) was detected by western blot in whole cell lysates of mock-infected Hela cells or HeLa cells infected with the indicated (recombinant) viruses at 7 and 10 hpi. HSP90 was detected as loading control. Representative images of n = 3 independent experiments are shown. (B-G) EMCV-Wt (B-D) or EMCV-2Apro (E-G) infected cells were treated with 10 μM of the P38 inhibitor SB203580 (SB) 1 hpi. (B,E) Intracellular and EV-enclosed virus titers 7 hpi were determined using end-point dilution assay. (C,F) Quantification of the fold change in EV-enclosed virus titers upon treatment with the P38 inhibitor SB, after correction for differences in intracellular virus production in the corresponding samples. (D, G) EV numbers were quantified by high resolution flow cytometry. Depicted is the fold increase in EV numbers during infection relative to the respective mock-infected controls. (H-J) Experiments indicated above were repeated for CVB3-Wt infected cells treated with 50 μM SB starting 30 min pre-infection. * p<0.05, ** p<0.005, ***p<0.0005, ****p<0.0001 as determined by a paired two-tailed t-test or one-sample t-test. Bar graphs display mean ±SD of n = 3–5 independent samples.