Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis

Figures

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  Figure 1. Schematic diagram of the PCV2 capsid protein sequence with the NLS region and predicted cleavage site (red arrow) indicated. The sequence of the predicted MLS used in this study is shown below, where an α-helix is marked in orange.

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  Figure 2. Subcellular localization of PCV2 Cap and mutations. (A) The sequence information of recombinant protein Cap-EGFP and mutations. (B) Confocal images of the over-expressed Cap(1–64)-EGFP, Cap(1–41)-EGFP, Cap(42–64)-EGFP, Cap(1–22)-EGFP, Cap(16–41)-EGFP and EGFP in PK15 cells. EGFP signal was indicated in green in PK15 cells. Scale bar: 10 μm. (C) Co-localization of Cap(1–22)-EGFP (green) and FBL (red) in PK15 cells. (D) Co-localization of Cap(16–41)-EGFP (green) and TXN2-DsRed (red) in PK15 cells.

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  Figure 3. Apoptosis of PCV2 Cap and Cap-DMLS in PK15 cells was analyzed by flow cytometry. PK-15 cells were transfected with recombinant plasmids of Cap and Cap–DMLS for 24 h. Precipitated cells were acquired by centrifugation, stained with Annexin-V/ propidium iodide (PI) staining kit and detected using flow cytometry. (A) Scatter plot of representative flow cytometry of Annexin-FITC (X-axis) and propidium iodide (PI) (Y-axis). Cells expressing Cap (left panel), cells expressing Cap-DMLS (middle panel), and cells with no DNA transfected as control (right panel). (B) Bar graph presented the average quantitative results of three independent flow cytometry experiments in PK15 cells (*** p < 0.001).

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  Figure 4. PCV2 wild-type virus and mutant infectious DNA clone were rescued. PK15 cells were transfected with various PCV2 infectious clone DNA (WT and DMLS mutant), and then the cells grew for more than 8 passages. (A) The confocal images of PK15 cells transfected with various PCV2 infectious clone DNA of WT (positive control, PC) and mutant, and immunofluorescence staining of PCV2 Cap. The infectious clone DNA containing only one copy of stem loop was transfected as negative control (NC). Green signal indicated the Cap and blue signal indicated the nucleus. Scale bar: 100 μm. (B) PCV2 genomic copies in each passage were detected by quantitative real-time PCR. The horizontal axis represented the numbers of cell passages, whereas the vertical axis represented the viral genomic copies. The results of three independent experiments were used to analyze.