PCV2 Cap protein nuclear import via importin α/β receptor: molecular insights and antiviral potential

Figures

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  Figure 1. The nuclear import mechanism of C3-GST-Cap. (A) PK15 cells co-transfected with plasmids pEGFP-Cap-GST with pmCherry-C1, pmCherry-C1-Ran, pmCherry-C1-RanQ69L, pmCherry-C1-M9M, pmCherry-C1-Bimax2 for 24 h. Green fluorescence indicates cells expressing pEGFP-Cap-GST, while red fluorescence signifies cells expressing pmCherry fusion proteins. All photomicrographs were captured at a magnification of 400×, with a scale bar of 50 μM. (B) The Cap fluorescence ratio of nucleus to cytoplasm (N/C) was calculated for the selected regions of interest basing on each confocal microscopy image by ImageJ software, and 40 cells were chosen for statistical analysis under each condition, Data are presented as means ± SEM of three independent biological experiments. Statistical analysis was conducted using Student’s t-test. (C) The graph illustrates the percentages of cells with exclusive nuclear (N) distribution or with both cytoplasmic and nuclear (C + N) distribution of pEGFP-Cap-GST, based on the distribution of Cap in various treatment groups as shown in panel (A).

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  Figure 2. The nuclear import mechanism of Myc-Cap. (A) PK15 cells co-transfected with plasmids Myc-Cap with pmCherry-C1, pmCherry-C1-Ran, pmCherry-C1-RanQ69L, pmCherry-C1-M9M, pmCherry-C1-Bimax2 for 24 h. The resultant cells were fixed, incubated with mouse anti-Cap antibody, and then stained with anti-mouse FITC. Green fluorescence indicates cells expressing Myc-Cap, while red fluorescence signifies cells expressing pmCherry fusion proteins. All images were captured using the parameters specified in Figure 1. Scale bar, 50 μm. (B) The fluorescence ratio of nucleus to cytoplasm (N/C) was measured utilizing the same methodology detailed in Figure 1B, N = 40 cells for each condition. Data are presented as means ± SEM of three independent biological experiments. (C) The graph illustrates the percentages of cells with exclusive nuclear (N) distribution or with both cytoplasmic and nuclear (C + N) distribution of Myc-Cap, based on the distribution of Cap in various treatment groups as shown in panel (A).

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  Figure 3. Verification of the interactions between Cap and importin α subunits. (A) Plasmids encoding Myc-Cap were co-transfected with the plasmids expressing seven Flag-tagged importin α subunits in 293 T cells for 36 h. (B) plasmids encoding pEGFP-C3-Cap were co-transfected with the plasmids expressing seven importin α subunits in 293 T cells for 36 h. Cell lysates were immunoprecipitated using Flag-agarose beads and the Cap protein was detected using anti-Myc pAbs or anti-GFP pAbs.

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  Figure 4. Verification of the interactions between Cap and transport subunits. (A) Ten nuclear import receptors were co-transfected with the Myc-Cap expression plasmid into 293 T cells for 36 h. (B) Additionally, plasmids encoding pEGFP-C3-Cap were co-transfected with plasmids expressing 10 nuclear import receptors in 293 T cells for the same duration. Cell lysates were then collected, immunoprecipitated using Flag-agarose beads, and subsequently immunoblotted with anti-Flag mAb, anti-Myc polyclonal antibody (pAb), and anti-GFP pAb.

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  Figure 5. Importazole (IPZ) blocks Importin-β-dependent nuclear localization of Cap and inhibits the production of infectious PCV2 progeny. (A) The viability of PK15 cells pretreated with 10, 20, or 40 μM of importazole was analyzed using a CCK-8 assay. (B) Confocal dish-cultured PK15 cells were transfected with pEGFP-GST-Cap or Myc-Cap. At 8 h post-transfection, cells were treated with dimethyl sulfoxide (DMSO) or IPZ (20 μM). The cells were fixed at 24 h post-transfection. The distribution of Cap was detected with an anti-Cap antibody and observed by confocal microscopy. Scale bar, 50 μm. (C) The methodology used in this panel was the same as Figures 1B, 2B. (D) The graph depicts the percentages of cells exhibiting exclusive nuclear (N) localization or a combination of cytoplasmic and nuclear (C + N) localization of pEGFP-Cap-GST or Myc-Cap, based on the distribution of Cap within various treatment groups as depicted in panel (B). PK15 cells were infected with PCV2 at an MOI of 1 and treated with the indicated concentration of IPZ, cells were harvested at 24 hpi. (E) Viral protein synthesis was assayed by western blotting using antibodies against the Cap protein, with β-actin serving as…

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  Figure 6. Ivermectin (IVM) inhibits Imp-α-mediated nuclear localization of Cap but does not affect the production of infectious PCV2 progeny. (A) The viability of PK15 cells pretreated with 10, 15, or 25 μM of IVM was analyzed using a CCK-8 assay. (B) Confocal dish-cultured PK15 cells were transfected with pEGFP-Cap-GST or Myc-Cap. At 8 h post-transfection, cells were treated with dimethyl sulfoxide (DMSO) or IVM (15 μM). The cells were fixed at 24 h post-transfection. The distribution of Cap was detected with an anti-Cap antibody and observed by confocal microscopy. Scale bar, 50 μm. (C) The methodology used in this panel was the same as Figures 1B, 2B. (D) The graph shows the percentages of cells displaying exclusive nuclear (N) localization or a combination of cytoplasmic and nuclear (C + N) localization for pEGFP-Cap-GST or Myc-Cap. The classification is based on the distribution of Cap across various treatment groups, as depicted in Panel B. PK15 cells were infected with PCV2 at an MOI of 1 and treated with the indicated concentration of IVM, cells were harvested at 24 hpi. (E) Viral protein synthesis was assayed by western blotting (WB) using antibodies against the Cap protein, with…