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. 2012 Feb;86(3):1328-38.
doi: 10.1128/JVI.06104-11. Epub 2011 Nov 23.

Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5

Zhonghua Li  1 , Yongqiang Wang, Yanfei Xue, Xiaoqi Li, Hong Cao, Shijun J Zheng
Affiliations

Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5

Zhonghua Li et al. J Virol. 2012 Feb.

Abstract

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although IBDV-induced host cell apoptosis has been established, the underlying molecular mechanism is still unclear. We report here that IBDV viral protein 5 (VP5) is a major apoptosis inducer in DF-1 cells by interacting with the voltage-dependent anion channel 2 (VDAC2) in the mitochondrion. We found that in DF-1 cells, VP5-induced apoptosis can be completely abolished by 4,4'-diisothiocyanatostibene-2,2'-disulfonic acid (DIDS), an inhibitor of VDAC. Furthermore, knockdown of VDAC2 by small interfering RNA markedly inhibits IBDV-induced apoptosis associated with decreased caspase-9 and -3 activation and cytochrome c release, leading to increased IBDV growth in host cells. Thus, VP5-induced apoptosis during IBDV infection is mediated by interacting with VDAC2, a protein that appears to restrict viral replication via induction of cell death.

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Figures

Fig 1
Fig 1
Induction of DF-1 cell death by the viral components of IBDV. (A) IBDV infection caused a CPE in DF-1 cells. DF-1 cells were mock infected or infected with IBDV at an MOI of 10. Forty-eight hours postinfection, cells were observed by phase-contrast microscopy. (B) Flow cytometry analysis of IBDV-induced cell death in DF-1 cells. Thirty-six hours postinfection, cells were harvested and stained with annexin V-FITC and analyzed by flow cytometry. Mock-infected cells were used as controls. (C) Determination of cell death in IBDV-infected DF-1 cells by trypan blue dye exclusion assay. DF-1 cells were infected as described for panel A. Forty-eight hours postinfection, dead cells were counted under a microscope after trypan blue dye staining. Data are representative of three independent experiments. (D and E) Activities of caspase-3 (D) and caspase-9 (E) in DF-1 cells after IBDV infection. DF-1 cells were infected as described for panel A. Twenty hours after infection, the enzymatic activities of caspase-3 and caspase-9 were examined as described in Materials and Methods. Graphs show means ± SD (n = 3). ***, P < 0.001. (F) Expression of GFP-VP2, -VP3, -VP4, or -VP5 fusion proteins in DF-1 cells. DF-1 cells (6 × 105) were seeded on six-well plates and cultured overnight. Cells were transfected with pEGFP-C1, pEGFP-VP2, pEGFP-VP3, pEGFP-VP4, or pEGFP-VP5 plasmid. Twenty-four hours after transfection, cell lysates were prepared and examined with Western blotting (WB) using anti-GFP antibodies. (G) Flow cytometry analysis of apoptosis in DF-1 cells transfected to express GFP, GFP-VP2, GFP-VP3, GFP-VP4, or GFP-VP5 fusion proteins. DF-1 cells were transfected with different plasmids as described for panel F. Twenty-four hours after transfection, cells were harvested, stained with PI, and analyzed by flow cytometry. GFP-positive cells were gated for further analysis of PI staining-positive cells. Data are representative of three independent experiments.
Fig 2
Fig 2
Interaction of VP5 with VDAC2. (A to C) Interaction of IBDV VP5 with exogenous VDAC2. HEK293T cells (A) and DF-1 cells (B) were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted with anti-FLAG or anti-Myc antibodies. (C) Cell lysates were prepared as described above, immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-FLAG or anti-Myc antibodies. (D) Interaction of VP5 with endogenous VDAC2. HEK293T cells or DF-1 cells were transfected with pRK5-FLAG-VP5 or empty vector as a control. Thirty-six hours after transfection, cell lysates were prepared and immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG or anti-VDAC2 antibodies.
Fig 3
Fig 3
The portion of VP5 from amino acids 1 to 50 is responsible for binding to VDAC2. (A) Schematics represent the genes encoding the full-length VP5 and truncated VP5 molecules (Δ1 through Δ4). The numbers indicate the amino acid positions in the molecule. (B) Endogenous VDAC2 interacted with different truncated VP5 proteins. HEK293T cells (4 × 105) were transfected with full-length Myc-VP5 (wild type [WT]) and different truncated Myc-VP5 molecules (Δ1, aa 50 to 145; Δ2, aa 100 to 145; Δ3, aa 1 to 100; Δ4, aa 1 to 50) or empty vectors. Thirty-six hours after transfection, cell lysates were prepared and immunoprecipitated with anti-Myc monoclonal antibody. The pellets were examined by Western blotting using anti-VDAC2 monoclonal antibody. (C) Schematic representing the binding domain (amino acids 1 to 50) of VP5 for VDAC2.
Fig 4
Fig 4
Colocalization of VP5 with VDAC2 in the mitochondrion. (A to C) Localization of exogenous VP5 and VDAC2. HEK293T cells (2 × 105) were seeded on 24-well plates with coverslips in the wells and cultured overnight. Cells were cotransfected with pDsRed-VP5 and pEGFP-VDAC2. Twenty-four hours after transfection, cells were fixed with 1% paraformaldehyde. After washes, the cell nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. (D to F) Colocalization of VP5 with endogenous VDAC2. HEK293T cells were transfected with pEGFP-VP5 plasmid. Twenty-four hours after transfection, cells were fixed with 1% paraformaldehyde. After washes, the fixed cells were permeabilized with 0.1% Triton X-100 and immunostained with anti-VDAC2 and TRITC-conjugated secondary antibodies. Nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. (G to L) Colocalization of IBDV VP5 with endogenous VDAC2 in IBDV-infected cells. HEK293T cells were mock infected or infected with IBDV at an MOI of 10. Twenty-four hours after infection, cells were fixed and probed with mouse anti-VP5 antiserum antibody and rabbit anti-VDAC2 antibodies, followed by the FITC-conjugated goat antimouse antibody (green) and TRITC-conjugated goat antirabbit antibody (red). Nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. (M to R) Colocalization of VP5 with VDAC2 in the mitochondrion. HEK293T cells were transfected with pEGFP-VDAC2 or pEGFP-VP5 plasmids. Twenty-four hours after transfection, cells were stained by MitoTracker Red for the mitochondrion and observed under a laser confocal scanning microscope.
Fig 5
Fig 5
Inhibited VP5- or IBDV-induced apoptosis and restricted viral release in DF-1 cells treated by the VDAC inhibitor DIDS. (A) Inhibition of IBDV VP5-induced apoptosis in DF-1 cells by DIDS. DF-1 cells (6 × 105) were seeded on six-well plates and cultured overnight. Cells were transfected with pEGFP-C1 or pEGFP-VP5 plasmids by Lipofectamine LTX. Six hours after transfection, cells were cultured in the presence of 10 μΜ DIDS in dimethyl sulfoxide (DMSO; final concentration, <1% [vol/vol]) for 3 h. Three hours after incubation, medium was changed with fresh DMEM. Twenty-four hours after transfection, cells were harvested and stained with PI, followed by flow cytometry analysis. GFP-positive cells were gated for the further analysis of PI staining-positive cells. (B) Percentage of apoptotic cells transfected with pEGFP-C1 or pEGFP-VP5 with or without 10 μΜ DIDS. (C) Inhibition of IBDV-induced apoptosis in DF-1 cells by DIDS. DF-1 cells (6 × 105) were seeded on six-well plates and cultured overnight. Cells were mock infected or infected with IBDV at an MOI of 10 at 37°C for 3 h and then washed with PBS before incubation with DIDS (100 μΜ) or dimethyl sulfoxide for 3 h. Cells were again washed with PBS, followed by incubation with 10% DMEM. Thirty-six hours postinfection, cells were harvested and stained with annexin V-FITC and analyzed by flow cytometry. (D) Percentage of apoptotic cells after mock or IBDV infection in the presence of DIDS or dimethyl sulfoxide as a control. Data are representative of three independent experiments. (E and F) Effects of DIDS on IBDV growth and viral release. DF-1 cells were infected with IBDV (MOI = 10). Three hours after infection, cells were washed with PBS and then incubated with 100 μM DIDS or dimethyl sulfoxide for another 3 h. Cells were again washed with PBS, followed by incubation with 10% DMEM. At different time points (12, 24, 48, and 72 h) after IBDV infection, the viral loads in the cell cultures (E) and the supernatants (F) were determined by TCID50 determination using 96-well plates. The significance of the difference between DIDS-treated cells and controls was performed by ANOVA (P < 0.05). The graph shows the average of the viral loads in DF-1 cells from three individual experiments.
Fig 6
Fig 6
Knockdown of VDAC2 inhibits IBDV-induced apoptosis. (A) Effects of VDAC2 RNAi on the expression of endogenous VDAC2. DF-1 cells (4 × 105) were transfected with siRNA (RNAi#1 to RNAi#3) or controls as described in Materials and Methods. Forty-eight hours after the second transfection, cell lysates were prepared and examined by Western blotting with anti-VDAC2 antibody. Endogenous β-actin expression was used as an internal control. (B) Relative levels of VDAC2 in VDAC2 RNAi-treated cells. The density of bands in panel A was quantitated by densitometry. The relative levels of VDAC2 were calculated as follows: band density of VDAC2/band density of β-actin. NC, nontreated control. (C to F) Representative morphological changes of DF-1 cells after infection with IBDV or mock infection. VDAC2 RNAi cells (E), control RNAi cells (F), and normal DF-1 cells (D) were infected with IBDV at an MOI of 10. Thirty-six hours after infection with IBDV, cells were examined by phase-contrast microscopy. Mock-infected normal DF-1 cells were used as a control (C). Magnification, ×200. (G to I) Knockdown of VDAC2 inhibited IBDV-induced apoptosis. Normal DF-1 cells (G), control RNAi cells (H), and VDAC2 RNAi cells (I) were mock infected or infected with IBDV at an MOI of 10. Thirty-six hours postinfection, cells were harvested and stained with annexin V-FITC and PI, followed by flow cytometry analysis. (J) Percentage of surviving DF-1 cells, control (Con) RNAi cells, and VDAC2 RNAi cells with mock or IBDV infection. Data are representative of three independent experiments.
Fig 7
Fig 7
Knockdown of VDAC2 inhibited IBDV-induced activation of caspase-9 and -3 and release of cytochrome c and enhanced IBDV growth. (A and B) Normal DF-1 cells, control RNAi cells, and VDAC2 RNAi cells were mock infected or infected with IBDV at an MOI of 10. Twenty hours postinfection, the enzymatic activities of caspase-9 and -3 were examined as described in Materials and Methods. (C) Western blot analysis of cytochrome c (14 kDa) and β-actin (42 kDa) in cytosolic fractions prepared from normal DF-1 cells, RNAi control cells, and VDAC2 RNAi cells mock infected or infected with IBDV at an MOI of 10 for 24 h. Data are representative of three independent experiments. (D and E) Mock-transfected cells, control RNAi cells, and VDAC2 RNAi cells were infected with IBDV at an MOI of 10. At different time points (12, 24, 48, and 72 h) after IBDV infection, the viral titers in the cell cultures (D) or supernatants (E) were determined by TCID50 analysis using 96-well plates. The significance of the difference between VDAC2 RNAi and control RNAi treatments was performed by ANOVA (P < 0.001). The graph shows the average of viral titers in DF-1 cells from three individual experiments.
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