The serine-48 residue of nucleolar phosphoprotein nucleophosmin-1 plays critical role in subcellular localization and interaction with porcine circovirus type 3 capsid protein

Figures

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  Figure 1. NPM1 knockdown abolished the nucleolar localization of PCV3 Cap. A NPM1-knocked down and shCON-transfected PK-15 cells were respectively transfected with mCherry-PCV3-Cap for 24 h, and cells were fixed and then subjected to confocal microscopy analysis. GFP signals indicate silenced PK-15 cells. Nuclei were stained with 4′, 6′-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. B, C Images of PK-15 cell line expressing shRNA mediated by lentivirus (B), and the knockdown efficiency of NPM1 in shCON-transfected and NPM1-silenced PK-15 cells (C). The cell lysates were subjected to immunoblotting using the indicated antibodies. D The nuclear DNA content of shRNA-control and NPM1-silenced cells were measured using PI staining and FACS analysis to analyze the cell cycle. E The histogram was analyzed quantitatively by a curve-fitting program to determine the percentage of cells in the S phase of both cells. Data are presented as means ± SD of three independent biological experiments. ns not significant.

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  Figure 2. PCV3 Cap colocalizes and interacts with NPM1. A, B NPM1 colocalization with PCV3 Cap in transfected cells. HEK293T (A) and PK-15 (B) cells were cotransfected with GFP-PCV3-Cap and mCherry-NPM1 for 24 h, and cells were fixed and then subjected to confocal microscopy analysis. Nuclei were stained with 4′, 6′-diamidino-2-phenylindole (DAPI) (A, B). Scale bar, 10 μm. C. PK-15 cells were transfected with empty vector and Flag-PCV3-Cap for 48 h. D, E HEK293T cells were cotransfected with Myc-PCV3-Cap and Flag-NPM1 for 48 h. The cell lysates were immunoprecipitated with Flag beads (C, D) or anti-Myc purified IgG (E). F Lysates of Flag-PCV3-Cap transfected 293T cells mixed with the GST, GST-NPM1 proteins were pulled down with glutathione S-transferase (GST) beads, and then subjected to GST pull-down assays, followed by immunoblotting using corresponding antibodies.

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  Figure 3. Binding domain identification of PCV3 Cap with NPM1. A Schematic representation of the truncation mutants of PCV3 Cap used in this study. B, C The N-terminal nuclear localization signal of PCV3 Cap interacted with NPM1. HEK293T cells were cotransfected with plasmids encoding full-length PCV3 Cap or truncation mutants fused with a GFP-, or Flag-gst tag, along with Flag-NPM1; cell lysates were subjected to immunoprecipitation or GST pull-down and immunoblotting using the indicated antibodies. D, E The nuclear localization signals (NLSs) within capsid protein of porcine circovirus type 1, 2, 3, 4 and circoviruses from other species were responsible for the binding to NPM1. HEK293T cells were cotransfected with plasmids encoding NLSs of PCV1, 2, 3, 4 (D) and circoviruses from terrestrial, aquatic and avian species, including pigs, canaries, canines, minks, dragonflies, pigeons, ducks, bats, geese, and parrots (E), along with Flag-NPM1; cell lysates were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. F Identification of the nucleolar localization signal in PCV3 Cap. HEK293T cells were cotransfected with GFP-NCL and mCherry-PCV3-Cap-WT, -M1, or -M2…

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  Figure 4. Serine-48 of NPM1 is critical for binding to PCV3 Cap. A Schematic representation of the OligoD, HistonD, and NBD of NPM1 and their truncation mutants used in this study. B, C The OligoD of NPM1 (aa 1–117) interacted with PCV3 Cap. HEK293T cells were cotransfected with plasmid GFP-NPM1-WT or serial GFP-NPM1 truncated mutants D1 to D6, along with Flag-PCV3-Cap and Flag-gst-PCV3-Cap expression vectors. The cell lysates were subjected to immunoprecipitation or GST pull-down and immunoblotting using the indicated antibodies. D Prediction of binding pattern of N-terminal OligoD from NPM1 with NoLS of PCV3 Cap using PyMOL software. E The N-terminal amino acid sequence of the OligoD of NPM1. Serine (S) or threonine (T) residues are marked in red. F, G Mapping the crucial amino acids of OligoD responsible for binding to PCV3 Cap. HEK293T cells were cotransfected with NPM1 or NPM1 mutants (-S48A, -S48E, -S88A, -S88E, -T95A, or -T95D), along with Flag-PCV3-Cap and Flag-gst-PCV3-Cap expression vectors, and the cell lysates were subjected to immunoprecipitation or GST pull-down and immunoblotting using the indicated antibodies.

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  Figure 5. Charge property of serine-48 within NPM1 determines its subcellular localization and interaction with PCV3 Cap. A, B Charge property of serine-48 of NPM1 is responsible for binding to PCV3 Cap. HEK293T cells were cotransfected with NPM1 or NPM1 mutants (-S48E, -S48D, -S48A, -S48T, -S48K, or -S48R), along with Flag-PCV3-Cap and Flag-gst-PCV3-Cap expression vectors, and the cell lysates were subjected to immunoprecipitation or GST pull-down and immunoblotting using the indicated antibodies. C Nucleolin colocalization with NPM1 and various NPM1 mutants in transfected cells. HEK293T cells were cotransfected with GFP-NPM1 or NPM1 mutants (-S48E, -S48D, -S48A, -S48T, -S48K, or -S48R), along with mCherry-NCL for 24 h, and cells were fixed and then subjected to confocal microscopy analysis. Nuclei were stained with 4′, 6′-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm.