The Nucleolar Localization Signal of Porcine Circovirus Type 4 Capsid Protein Is Essential for Interaction With Serine-48 Residue of Nucleolar Phosphoprotein Nucleophosmin-1

Figures

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  FIGURE 1. The N-terminal residues 1–37 of PCV4 Cap are a nucleolar localization signal. (A) Schematic depicting the truncated mutants of PCV4 Cap in the context. (B) HEK293T cells were co-transfected with mCherry-NCL and GFP-PCV4-Cap-WT, Cap-M1, Cap-M2, Cap-M3, or Cap-M4 for 24 h. The HEK293T cells were incubated with DAPI and then observed under a confocal microscope. Scale bar, 10 μm.

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  FIGURE 2. PCV4 Cap binds to NPM1. (A,B) Colocalization of NPM1 with PCV4 Cap in co-transfected cells. HEK293T cells (A) or PK-15 cells (B) were co-transfected with GFP-PCV4-Cap and mCherry-NPM1 for 24 h, and the cells were fixed followed by nuclei staining with DAPI (A,B). Scale bar, 10 μm. (C) PK-15 cells were co-transfected with an empty vector and Flag-PCV4-Cap for 48 h. (D,E) The cell lysate extracts were immunoprecipitated with Flag beads (C,D) or anti-Myc purified IgG (E). (F) The Flag-PCV4-Cap-transfected HEK293T cell lysates mixed with the GST, and GST-NPM1 proteins subjected to a GST pull-down assay and then detected by western blotting using corresponding antibodies.

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  FIGURE 3. N-terminal residues 1–20 of PCV4 Cap are essential for binding to NPM1. (A,B) HEK293T cells were co-transfected with plasmids containing full-length PCV4 Cap or truncated mutants, together with the Flag-NPM1 plasmid for 48 h; the cell lysate extracts were immunoprecipitated with Flag beads (A) or anti-GFP purified IgG (B) and then detected by western blotting using the indicated antibodies.

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  FIGURE 4. NPM1 serine-48 is crucial for binding to PCV4 Cap. (A,B) The NPM1-OligoD (1–117 aa) interacted with PCV4 Cap. HEK293T cells were co-transfected with expression plasmids GFP-NPM1-WT or its serial GFP-NPM1 truncated mutants M1 to M6, together with the Flag-PCV4-Cap or Flag-gst-PCV4-Cap plasmid. The cell lysate extracts were immunoprecipitated or subjected to a GST pull-down assay followed by western blotting using the indicated antibodies. (C,D) Mapping the critical amino acids of OligoD required for interaction with PCV4 Cap. HEK293T cells were co-transfected with NPM1 or NPM1 mutant plasmids (NPM1-S48A, NPM1-S48E, NPM1-S88A, NPM1-S88E, NPM1-T95A, or NPM1-T95D), together with the Flag-PCV4-Cap or Flag-gst-PCV4-Cap plasmid, and the cell lysate extracts were immunoprecipitated or subjected to a GST pull-down assay followed by western blotting using the indicated antibodies.

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  FIGURE 5. The serine-48 charge property of NPM1 determines its oligomerization and NPM1/Cap interaction. (A,B) The serine-48 charge property of NPM1 is essential for interaction with PCV4 Cap. HEK293T cells were co-transfected with NPM1 or NPM1 mutant plasmids (NPM1-S48A, NPM1-S48D, NPM1-S48E, NPM1-S48K, NPM1-S48R, or NPM1-S48T), together with the Flag-PCV4-Cap or Flag-gst-PCV4-Cap plasmid, and the cell lysate extracts were immunoprecipitated or subjected to GST pull-down assay followed by western blotting using the indicated antibodies. (C) Charged amino acids replaced by serine-48 disrupted the oligomerization of NPM1. Lysate extracts of PK-15 cells co-transfected with an empty vector, GFP-NPM1-WT, GFP-NPM1-S48A, GFP-NPM1-S48D, GFP-NPM1-S48E, GFP-NPM1-S48K, GFP-NPM1-S48R, or GFP-NPM1-S48T for 48 h were immunoprecipitated with anti-GFP purified IgG and then subjected to Co-IP assays.