Molecular characterization and antiapoptotic function analysis of the duck plague virus Us5 gene

Figures

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  Figure 1. The transcription and expression kinetics of the DPV Us5 gene. (A) Relative transcript levels of the DPV Us5 gene at 6, 12, 24, 36, 48, 54, and 60 hpi. (B) Western blotting analysis for Us5 and β-actin at 6, 12, 24, 36, 48, 54, and 60 hpi (approximately 130 kDa) with anti-Us5 serum and anti-β-actin serum.

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  Figure 2. Pharmaceutical identification of virion gene type. M: DL2000 marker; 1: DEV-infected cells treated with 50 μg/ml cycloheximide (CHX); 2: DEV-infected cells treated with 300 μg/mL ganciclovir (GCV); 3: DPV-infected cells without drugs; 4: untreated DEF cells. The DPV Us5 gene (156 bp) and the L gene Us2 (111 bp) are shown.

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  Figure 3. Dynamic intracellular localization of the Us5 protein during DPV infection at different times. Fluorescence microscopic images of Us5 localization (green in all images) at 6, 12, 24, 36, 48, 54 and 60 hpi. Nuclei are indicated in blue (DAPI) and cytoskeleton are indicated red (Phalloidin).

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  Figure 4. The Us5 protein is a component of the virion, as determined by Western blotting.

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  Figure 5. H2O2 induces apoptosis in DEF cells. (A) Cells were observed using phase contrast microscopy (×100). Non-H2O2-treated cells were used as controls (B). The cells were fixed and stained with DAPI. The arrows indicate apoptotic nuclei, and non-H2O2-treated cells were used as controls. (C) TUNEL assay. The arrows refer to TUNEL- positive cells, and non-H2O2-treated cells were used as controls.

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  Figure 6. Activity of caspase-3 and caspase-9 induced by H2O2 in DEF cells. (A) The mRNA expression levels of caspase-3 and caspase-9 in H2O2-treated and non-H2O2-treated cells. (B) The activity of caspase-3/7 and caspase-9 in H2O2-treated and non-H2O2-treated cells. All data are presented as the means ± SD from three individual experiments. Non-H2O2-treated cells were used as controls. p* < 0.05 compared to the non-H2O2-treated controls. (C) Flow Cytometry results indicated that H2O2-treated cells have 22% apoptosis cells.

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  Figure 7. Expression of the Us5 protein in DEF cells. (A) Indirect immunofluorescence of DPV Us5 in DEF cells. Costaining was performed with a rabbit anti-DPV-Us5 polyclonal antibody and a fluorescein-conjugated goat anti-rabbit IgG antibody. (B) Western blotting of DPV Us5 in DEF cells. A rabbit anti-DPV-Us5 polyclonal antibody was used as the primary antibody. A goat anti-rabbit IgG (H + L)-HRP antibody was used as the secondary antibody. Noninfected cells were used as controls. (C) Western blotting of pCAGGS-Us5 in DEF cells. A duck anti-DPV polyclonal antibody was used as the primary antibody. A goat anti-duck IgG (H + L)-HRP antibody was used as the secondary antibody. Cells transfected with the pCAGGS plasmid were used as the controls.

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  Figure 8. Effect of the DPV Us5 protein on H2O2-induced apoptosis. (A) TUNEL assay. The arrows indicate TUNEL-positive cells. Cells transfected with the pCAGGS plasmid and treated with H2O2 were used as controls. (B) The activity of caspase-3/7 and caspase-9 in cells transfected with the pCAGGS-Us5 plasmid and treated with H2O2 and cells transfected with the pCAGGS plasmid and treated with H2O2. (C) H2O2-treated cells transfected with the pCAGGS-Us5 plasmid cloud inhibit a lot of apoptosis. All data are presented as the means ± SD from three individual experiments. p* < 0.05, p** < 0.01 compared to the non-Us5-expressing control.

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  Figure 9. Anti-apoptosis ability of DPV CHv-BAC parental viruses, DPV CHv-BAC-ΔgJ mutant viruses and CHv-BAC-ΔgJR rescueviruses. (A) The apoptosis DEF cells of recombination viruses relative to the parental virus were investigated at 48 hpi utilizing DAPI staining. (B) The apoptosis DEF cells of recombination viruses relative to the parental viruses were investigated at 48 hpi utilizing Flow Cytometry. All data are presented as the means ± SD from three individual experiments. p* < 0.05 compared to the DPV CHv-BAC parental viruses and the DPV CHv-BAC-ΔgJR rescue viruses.