Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication

Figures

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  FIGURE 1. Gene ontology and KEGG pathway enrichment analysis of interactions. (A) Annotation of proteins interacting with PCV3 Cap using Gene Ontology. (B) KEGG pathway analysis of cellular proteins interacting with PCV3 Cap. (C) Enrichment of differential pathway terms in KEGG pathways. Cut-off P-value of 0.01 was used to reduce false discovery rate. (D) Top 20 KEGG pathways.

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  FIGURE 2. Mapping and identification of protein interactions. (A) Mapping of protein-protein interactions (PPIs) network using STRING database. Top ranking PPIs were selected to map the interaction network. The PPIs network with PCV3 Cap was constructed by Cytoscape software. (B) Confirmation of interaction between PCV3 Cap and NPM1. HEK293T cells were co-transfected with plasmids expressing FLAG-Cap and HA-NPM1. Co-transfection with an empty vector was used as a negative control. Cell lysates were prepared at 24 h after transfection and immunoprecipitated with HA beads. Protein samples were separated by SDS-PAGE and detected with antibodies against FLAG and Cap by western blotting. (C) Colocalization of PCV3 Cap with NPM1 and nucleolar proteins. PK15 cells were co-transfected with plasmids expressing HA-Cap with NPM1-GFP, nucleolin-GFP, and fibrillarin-GFP, respectively. The cells were incubated with the antibodies corresponding to the HA tag at 24 h post-transfection, and followed by the Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with DAPI (blue).

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  FIGURE 3. Determination of the binding domains of PCV3 Cap with NPM1. (A) Engineering of truncated constructs of PCV3 Cap, GFP-Cap (1–38), GFP-Cap (39–106), GFP-Cap (107–174), and GFP-Cap (175–214). (B) The interaction regions between NPM1 and Cap confirmed by Co-IP in HEK293FT cells with co-transfection. At 24 h post-transfection, the cell lysates were immunoprecipitated with Pierce Anti-HA Magnetic Beads, western blot analysis using antibodies to HA, GFP, and β-actin. (C) Colocalization analysis. PK15 cells were co-transfected with GFP-Cap, truncation constructs of GFP-Cap, and HA-NPM1. The cells were incubated with the antibodies corresponding to the HA tag at 24 h post-transfection, and followed by the Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with DAPI (blue).

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  FIGURE 4. Endogenous colocalization analysis. PCV3 Cap induced the translocation of NPM1 to the cytoplasm from the nucleus. (A,B) PK15 cells were transfected to express FLAG-Cap and GFP-Cap. The cells were incubated with the antibodies corresponding FLAG tag, NPM1, and NCL followed by the Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with DAPI (blue). (C) The cell nuclear and cytoplasmic extraction was performed after PK15 cells transfected GFP-Cap plasmid. At 48 h post-transfection, the protein samples were prepared and detected using antibodies against GFP and NPM1 by western blot analysis. Histone H3 and β-actin were used as fractionation quality controls. (D) The relative gray intensity of cytoplasmic NPM1 was calculated using ImageJ. Two-tailed Student’s t-test was used for statistical analysis. Each experiment was independently done three times. Data represent by mean ± standard deviation (SD). Asterisks (*) indicate the statistical significance: *P < 0.05; NS: not significant.

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  FIGURE 5. PCV3 increased the overall abundance of NPM1 in infected PK15 cells. (A) PK15 cells were infected with PCV3 and harvested at 24, 48, 72, and 96 h post-infection. The cell lysates were subjected to western blotting to examine the levels of Cap and NPM1 protein. β-actin was used as an internal control. (B) Graph showing the levels of NPM1 normalized against β-actin. The relative gray intensity was quantified using ImageJ. Each experiment was independently done three times. Data represent by the mean ± standard deviation (SD) (***P < 0.001; NS, not significant). (C) Real-time quantitative PCR was used to calculate the expression of NPM1 mRNA in PCV3-infected cells at 24, 48, 72, and 96 hpi. Expression was normalized to the GAPDH mRNA level. The relative expression was analyzed using GraphPad Prism. Each experiment was independently done three times. Data represent the mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.01; NS, not significant). (D) PK15 cells were infected with PCV3, fixed, and subjected to immunofluorescence assay at 0, 24, 48, 72, and 96 hpi, respectively. Immunofluorescence signals revealed the expression of Cap (green) and NPM1 protein (red). DAPI was used to stain cell…

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  FIGURE 6. Enhancement of PCV3 replication by NPM1 overexpression in lentivirus-transduced PK15 cells. (A) Lentiviruses were packed in HEK293FT cells, and the lentiviruses of NPM1-GFP and GFP transduced into PK15 cells were examined by microscopy. (B) The viability of lentivirus-transduced PK15 cells was analyzed by CCK-8 assay at 48, 72, and 96 h post-transduction. (C) Lentivirus-transduced PK15 cells and no-transduced PK15 cells were infected with PCV3. The infected cells were harvested at 48, 72, and 96 h post-infection. Western blotting was used to examine the expression levels of Cap, NPM1-GFP, GFP, and β-actin. (D) The copy numbers of viral DNA were determined by qPCR. Data represent the mean ± standard deviation (SD) of three independent experiments (**P < 0.01; NS, not significant).

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  FIGURE 7. Suppression of PCV3 proliferation by silencing NPM1 expression in PK15 cells. (A) Silencing efficiency of NPM1 expression with different concentrations of specific siRNA (siNPM1). PK15 cells were transfected with siRNA for NPM1 at a concentration of 20 or 40 pmol. siNC and untransfected cells were used as negative controls. (B) Viability of NPM1 gene-silenced PK15 cells. (C) PCV3 replication in NPM1 gene-silenced PK15 cells by transfecting siNPM1-577. Cells were infected with PCV3 and harvested at 48, 72, and 96 h post-infection. Western blot was exploited to determine the expression levels of Cap, NPM1, and β-actin. (D) The copy numbers of viral DNA were determined by qPCR in siNPM1-577-transfected PK15 cells. Data represent the mean ± standard deviation (SD) of three independent experiments (**P < 0.01; ***P < 0.001; NS, not significant). (E) NPM1 inhibitor NSC348884 and DMSO-treated cells were infected with PCV3 for 48, 72, and 96 h. Western blot was used to examine the expression levels of Cap, NPM1, and β-actin. (F) NPM1 expression in NSC348884- and DMSO-treated cells analyzed by immunofluorescence. (G) Viability of PK15 cells treated with NSC348884 and DMSO analyzed using a…