Enterovirus 71 non-structural protein 3A hijacks vacuolar protein sorting 25 to boost exosome biogenesis to facilitate viral replication

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  FIGURE 1. Enterovirus 71 (EV71) infection promotes exosome biogenesis. RD cells were mock-infected or infected with EV71 (MOI = 0.5). At 24 h post-infection, cell supernatants were harvested for exosome extraction. (A–C) The characteristics of exosomes were examined by transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) to observe the morphology (A), size and distribution (B), particles and protein concentration (C) of exosomes, respectively. The size is expressed by diameter. (D) The extracted exosomes were detected by Western blot analysis. HRS, Tsg101, CD9, and CD63 are used as exosome markers, and Calnexin is a marker of the cytosol to reflect cellular contamination. (E) The extracted EV71-induced exosomes were stained by Dil (red) and fluorophore-conjugated CD9 antibody (green), then examined by Leica TCS SP8 confocal laser scanning microscopy with Lightning mode. WCL, whole cell lysate. Graph shows as mean ± SD. *P < 0.05; ***P < 0.001.

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  FIGURE 2. Enterovirus 71 (EV71) 3A protein increases exosome biogenesis. (A) Schematic diagram of EV71 structure and non-structural proteins. (B–D) RD cells were transfected with 2 μg plasmids expressing HA-tagged EV71 non-structural proteins (B), or different doses (0, 0.5, 1, and 2 μg) of plasmids expressing GFP-tagged 3A or 3B protein (C), or different doses (0, 0.2, 0.5, 1, 1.5, and 2 μg) of plasmids expressing GFP-tagged 3A protein (D). At 24 h post-transfection, the extracted exosomes from cell supernatants and cell lysates were collected for Western blot analysis. HRS, Tsg101, CD9, and CD63 are used as exosome markers, while Calnexin is a marker of cytosol. β-actin is presented as an internal reference protein in cell lysates. WCL, whole cell lysate. #, the non-specific band. (E) RD cells were transfected with 2 μg plasmids expressing GFP or GFP-tagged 3A protein. At 24 h post-transfection, the extracted exosomes from cell supernatants were observed by TEM and measured by size and distribution, respectively.

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  FIGURE 3. Enterovirus 71 (EV71) 3A protein interacts with VPS25. (A) RD cells were transfected with plasmids expressing GFP or GFP-tagged 3A. Cell lysates were immunoprecipitated with anti-GFP (α-GFP) antibody. The protein samples were separated by SDS-PAGE and visualized with Coomassie blue staining. (B) HEK293T cells were co-transfected with empty or indicated plasmids expressing FLAG-tagged VPS25 or GFP-tagged 3A, followed by the immunoprecipitated with anti-FLAG (α-FLAG) or anti-GFP (α-GFP) antibody, respectively. The corresponding proteins were detected by Western blot analysis. (C) RD cells were infected with EV71 (MOI = 0.5) for 8 h. Immunofluorescence assay was performed after labeling proteins of EV71 3A (green), VPS25 (red), and staining nuclei with DAPI (blue). (D) Schematic of bimolecular fluorescence complementation assay (BiFc). (E–G) RD cells were co-transfected with Venus BiFc plasmids expressing control protein (VC155 or VN173), 3A protein (VC155-3A), and VPS25 protein (VN173-VPS25), respectively. At 24 h post-transfection, the expression of corresponding proteins was detected by Western blot analysis (E) or visualized by confocal microscopy (F). The fluorescence intensity…

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  FIGURE 4. The location of VPS25 in EV71-induced exosome biogenesis. (A–C) RD cells were mock-infected or infected with EV71 (MOI = 0.5) for 24 h (A,B). The cell images and count were captured (B). RD cells were infected with EV71 at different MOIs (C). At 24 h post-infection, extracted exosomes and cell lysates were analyzed by Western blot. (D) The extracted exosomes from panel (A) were detected by immunoelectron microscopy using anti-VPS25 antibody. (E) Schematic of immunocapture-based ELISA to detect protein on the surface of exosomes. (F) RD cells were mock-infected or infected with EV71 (MOI = 0.5). At 24 h post-infection, exosomes extracted from 10 mL of cell supernatant were detected by ELISA in the CD63 and VPS25 antibodies coating plates, respectively. Ab, antibody. Graph shows as mean ± SD. ns, not-significant. **P < 0.01.

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  FIGURE 5. Vacuolar protein sorting 25 (VPS25) mediates EV71-induced exosome biogenesis. (A,B) RD cells were transfected with 100 nM siRNA negative control (siNC), or against VPS25 (siVPS25-1#, -2#), respectively. At 48 h post-transfection, the level of VPS25 protein was measured by Western blot (A). Until 72 h post-transfection, the cell viability was measured using the CCK8 assay (B). (C,D) RD cells were transfected with siNC or siVPS25-1# for 24 h and then transfected with plasmids expressing GFP or GFP-tagged 3A (C), or infected with EV71 (MOI = 0.5) (D) for another 24 h. The extracted exosomes and cell lysates were analyzed by Western blot. The equal volume of exosomes in PBS of each sample with a total protein amount ranging from 0.5 to 2.0 μg was loaded. WCL, whole cell lysate.

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  FIGURE 6. Vacuolar protein sorting 25 (VPS25) mediates EV71-induced exosome to facilitate viral replication. (A) Schematic of kinetic time points in the EV71 life cycle. (B–D) RD cells were transfected with 100 nM siNC or siVPS25-1# for 24 h and then infected with EV71 (MOI = 0.5). At 8 and 12 h p.i., the cell lysates were analyzed by Western blot (B). At 24 h p.i., the cell lysates were analyzed by Western blot (C) and the progeny virus titer was measured by TCID50 assay (D). (E) RD cells were transfected with different doses of siNC or siVPS25-1# (0, 50, 100, and 150 nM) for 24 h and then infected with EV71 (MOI = 0.5) for another 24 h. Cell viability was measured using the CCK8 assay. Graph shows as mean ± SD. **P < 0.01.

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  FIGURE 7. GW4869 inhibits viral replication via reducing exosome biogenesis. (A) GW4869 acts as an exosome inhibitor in the endocytic pathway. (B–F) RD cells were incubated with GW4869 (20 μM) for 2 h and then infected with EV71 (MOI = 0.5). At 24 h p.i., the extracted exosomes and cell lysates were analyzed by Western blot (B). At 8 and 12 h p.i., the cell lysates were analyzed by Western blot (C). At 24 h p.i., cell lysates were analyzed by Western blot (D) and progeny virus titer was measured by TCID50 assay (E). WCL, whole cell lysate. (F) RD cells were incubated with different doses of DMSO or GW4869 (0, 10, 20, 40, and 50 μM) for 2 h and then infected with EV71 (MOI = 0.5) for another 24 h. Cell viability was measured using the CCK8 assay. Graph shows as mean ± SD. **P < 0.01.

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  FIGURE 8. A proposed mechanism underlying VPS25 mediates EV71-induced exosome biogenesis to facilitate viral replication. Upon EV71 infection, viral single positive-sense RNA utilizes host translation machinery to translate a polyprotein, which is cleaved into structural and non-structural protein. Among viral non-structural proteins, EV71 3A protein physically interacts with VPS25, a component of the ESCRT-II complex, which participates in MVB generation and exosome biogenesis. The release of progeny EV71 could be in the forms of lytical and non-lytical manners. VPS25-mediated exosomes are engaged in the EV71 transmission and infection, thereby facilitating EV71 replication.