Enteroviral 2B Interacts with VDAC3 to Regulate Reactive Oxygen Species Generation That Is Essential to Viral Replication

Figures

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  Figure 1. Mitochondrial ROS generation essential to EV71 replication. (A) Vero cells were mock infected (Mock) or infected (EV71) with BrCr virus at an MOI of 1.25, and stained with CellROX Deep Red for quantification of total cellular ROS (Total) or with MitoSOX Red for quantification of mitochondrial ROS (Mito) at 24 h post-infection. The MFI is expressed as fold change relative to that of mock-infected cells. The results are mean ± SD, n = 6. * p < 0.05, *** p < 0.005 vs. mock-infected cells. (B) Vero cells were mock infected (Mock) or infected (EV71) with BrCr virus at an MOI of 1.25; treated without or with 100 μM and 200 μM Mito-TEMPO; and stained with MitoSOX Red at 24 h post-infection. The MFI is expressed as fold change relative to that of untreated mock-infected cells. The results are mean ± SD, n = 6. * p < 0.05, ** p < 0.01 vs. mock-infected cells. (C) Cells were mock infected (Mock) or infected (EV71) with BrCr virus at an MOI of 1.25; treated without or with 100 μM and 200 μM Mito-TEMPO; and extracted for determination of EV71 genomic RNA. The RNA level is expressed as the percentage of that of untreated infected cells. The results are mean ± SD, n = 6. *** p < 0.005 vs.…

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  Figure 2. Identification of the 2B-interacting proteins through immunoprecipitation-mass spectrometry. (A) Vero cells were transfected with vectors encoding GFP-Myc (control) or GFP-2B-Myc proteins, and lysates were prepared. Immunoprecipitation with the anti-Myc antibody was followed by tryptic digestion and labeling with light (hydrogen) and heavy (deuterium) stable-isotope dimethyl formaldehyde. Peptides were analyzed by the strong cation exchange (SCX)/reverse-phase nanoscale liquid chromatography (LC) coupled with Orbitrap Elite hybrid mass spectrometer. The proteins with heavy/light ratio ≥ 10 and heavy/light count ≥ 2 were identified, and analyzed using IPA. (B) Cell lysate (Lysate) and the anti-Myc (Anti-Myc) immunoprecipitate (IP) were analyzed by Western blotting (WB) for the presence of GFP-Myc, GFP-2B-Myc and actin. The asterisk (*) indicates a band cross-reactive with the particular batch of anti-actin antibody. A representative result of three experiments is shown here. (C) The proteome dataset was analyzed using IPA, and the top canonical pathways are shown. The horizontal line shows the threshold for p-value. The bars are color coded according to their z-score or the…

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  Figure 3. Interaction between 2B and VDAC3. (A) Vero cells were transfected with GFP-2B-Myc expression vector (or the one expressing the control protein GFP-Myc) and N-FLAG-VDAC3 expression vector. Cell lysate was prepared for immunoprecipitation (IP) with anti-Myc (left panel) or anti-FLAG (right panel) antibody. The cell lysate and immunoprecipitate were analyzed by Western blotting with antibody to anti-Myc epitope tag, anti-FLAG epitope tag, or anti-actin (for total cell lysate). (B) Vero cells were transfected with N-FLAG-VDAC3 expression vector (or the expression vector encoding the FLAG-tagged C34V control protein) in addition to GFP-2B-Myc expression vector. Cells were lysed for immunoprecipitation (IP) with anti-FLAG (left panel) or anti-Myc (right panel) antibody. The cell lysate and immunoprecipitate were analyzed by Western blotting with antibody to anti-Myc epitope tag, anti-FLAG epitope tag, or anti-actin (for total cell lysate). The asterisk (*) indicates a degradation product of C34V. A representative result of three experiments is shown here.

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  Figure 4. Localization of 2B and VDAC3 to the periphery of mitochondria. (A–D) Vero cells were transfected with the GFP-2B expression vector, and stained with MitoTracker Red and Hoechst dye 33342 at 24 h post-transfection. They were counterstained with Hoechst 33342. The samples were processed for confocal microscopic examination of 2B (Green Channel), mitochondria (Red Channel), and nuclei (Hoechst 33342). The images are overlaid (Merged). (E–H) Vero cells were transfected with N-FLAG-VDAC3 expression vector, and stained with FITC-conjugated anti-FLAG antibody and MitoTracker Red at 24 h post-transfection. They were counterstained with Hoechst 33342. The samples were processed for confocal microscopic examination of FITC-conjugated anti-FLAG antibody-bound VDAC3 (Green Channel), mitochondria (Red Channel), and nuclei (Hoechst 33342). The images are overlaid (Merged). (I–L) Vero cells were transfected with GFP-2B and N-FLAG-VDAC3 expression vectors, and stained with Cy3-conjugated anti-FLAG antibody at 24 h post-transfection. They were counterstained with Hoechst 33342. The samples were processed for confocal microscopic examination of 2B (Green Channel), VDAC3 (Red Channel), and nuclei…

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  Figure 5. VDAC3 plays an essential role in viral replication. Vero and RD cells were transduced with the lentiviral vectors encoding control shRNA (NTC), or the shRNA against VDAC3 (shVDAC3), to generate Vero-NTC, Vero-shVDAC3, RD-NTC, and RD-shVDAC3 cells, respectively. (A–D) Vero-shVDAC3 and Vero-NTC cells were infected with EV71 at an MOI of 1.25 for 1 h (A,B), while RD-shVDAC3 and RD-NTC cells were infected at MOI of 0.01 (C,D). Twenty-four hours later, the titer (A,C) and genomic RNA level (B,D) of the progeny virus were determined. (E–H) Vero-shVDAC3 and Vero-NTC cells were infected with EV71 at an MOI of 10 for 1 h (E,F). RD-shVDAC3 and RD-NTC cells were similarly infected (G,H). Six hours later, the titer (E,G) and genomic RNA level (F,H) of the progeny virus were determined. (I,J) The levels of VDAC3 transcript in Vero-shVDAC3 and Vero-NTC cells (I), and RD-shVDAC3 and RD-NTC cells (J) were quantified by RT-qPCR. The level of VDAC3 transcript is expressed as the percentage of that of respective NTC cells. The results are mean ± SD, n = 6. * p < 0.05, ** p < 0.01, *** p < 0.005 vs. respective NTC cells.

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  Figure 6. VDAC3 is essential to mitochondrial ROS generation. (A–D) Vero-shVDAC3 and Vero-NTC cells (A,B) were mock infected or infected with EV71 virus at an MOI of 1.25. RD-shVDAC3, and RD-NTC cells (C,D) were mock infected or infected with EV71 virus at an MOI of 0.01. Twenty-four hours later, the cells were stained with CellROX Deep Red (A,C) for quantification of total cellular ROS, or with MitoSOX Red (B,D) for quantification of mitochondrial ROS. (E–H) Vero-shVDAC3 and Vero-NTC cells (E,F), or RD-shVDAC3 and RD-NTC cells (G,H) were mock infected or infected with EV71 virus at an MOI of 10. Six hours later, the cells were stained with CellROX Deep Red (E,G) for quantification of total cellular ROS or with MitoSOX Red (F,H) for quantification of mitochondrial ROS. The MFI is expressed as fold change relative to that of mock-infected NTC cells. The results are mean ± SD, n = 6. * p < 0.05, ** p < 0.01 vs. mock-infected NTC cells; § p < 0.05 vs. infected NTC cells.

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  Figure 7. 2B interacts with VDAC3 to induce mitochondrial ROS generation. (A) Vero cells were transfected with the GFPi-2B-Myc or GFPi-Myc expression vector. Forty-eight hours later, the cells were stained with MitoSOX Red for quantification of mitochondrial ROS. The results are mean ± SD, n = 6. ** p < 0.01 vs. the GFPi-Myc-expressing cells. (B) Vero cells were transfected as described above, and treated without or with 100 μM and 200 μM Mito-TEMPO at 24 h after transfection. Twenty-four hours later, the cells were stained with MitoSOX Red for quantification of mitochondrial ROS. The results are mean ± SD, n = 6. * p < 0.05, ** p < 0.01 vs. corresponding untreated cells. (C) Vero-shVDAC3 and Vero-NTC cells were transfected with the GFPi-2B-Myc or GFPi-Myc expression vector. Forty-eight hours later, the cells were stained with MitoSOX Red for quantification of mitochondrial ROS. The results are mean ± SD, n = 6. *** p < 0.01 vs. Vero-NTC cells; § p < 0.05 vs. the GFPi-2B-Myc-expressing Vero-NTC cells.

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  Figure 8. Alterations in antioxidant metabolism in EV71-infected cells. Vero-NTC and Vero-shVDAC3 cells were mock infected (Mock) or infected (EV71) at an MOI of 1.25, and extracted at 16 h post-infection for metabolomic analysis with UPLC-Q-TOF-MS (n = 18). Their GSSG/GSH ratio (A) and intracellular levels of 3-sulfinoalanine (B), taurine (C), hypotaurine (D) and 6-PG (E) are shown. The GSSG/GSH ratio and the levels of metabolites are expressed as a percentage of those of the mock-infected Vero-NTC cells. Data are mean ± SD (n = 18). * p < 0.05, ** p < 0.01, *** p < 0.005, vs. mock-infected Vero-NTC cells; § p < 0.05 vs. infected Vero-NTC cells.