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

Abstract

Thus far, there have been no reports on the molecular characterization and antiapoptotic function of the DPV Us5 gene. To perform molecular characterization of DPV Us5, RT-PCR and pharmacological inhibition tests were used to ascertain the kinetic class of the Us5 gene. Western blotting and an indirect immunofluorescence assay (IFA) were used to analyze the expression level and subcellular localization of Us5 in infected cells at different time points. Us5 in purified DPV virions was identified by mass spectrometry. The results of RT-PCR, Western blotting, and pharmacological inhibition tests revealed that Us5 is transcribed mainly in the late stage of viral replication. The IFA results revealed that Us5 was localized throughout DPV-infected cells but was localized only to the cytoplasm of transfected cells. Mass spectrometry and Western blot analysis showed that Us5 was a virion component. Next, to study the antiapoptotic function of DPV Us5, we found that DPV CHv without gJ could induce more apoptosis cells than DPV-CHv BAC and rescue virus. we constructed a model of apoptosis in duck embryo fibroblasts (DEFs) induced by hydrogen peroxide (H₂O₂). Transfected cells expressing the Us5 gene were protected from apoptosis induced by H₂O₂, as measured by a TUNEL assay, a caspase activation assay and Flow Cytometry assay. The TUNEL assay and Flow Cytometry assay results showed that the recombinant plasmid pCAGGS-Us5 could inhibit apoptosis induced by H₂O₂ in DEF cells. However, caspase-3/7 and caspase-9 protein activity upregulated by H₂O₂ was significantly reduced in cells expressing the recombinant plasmid pCAGGS-Us5. Overall, these results show that the DPV Us5 gene is a late gene and that the Us5 protein is a component of the virion, is localized in the cytoplasm, and can inhibit apoptosis induced by H₂O₂ in DEF cells.

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.

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.

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).

Figure 4

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

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.

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.

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.

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.

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.