Contribution of DEAD-Box RNA Helicase 21 to the Nucleolar Localization of Porcine Circovirus Type 4 Capsid Protein

Figures

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  FIGURE 1. DDX21 relocates from the nucleolus to the cytoplasm induced by PCV4 Cap overexpression. (A) Immunofluorescence analysis of DDX21 localization during PCV4 Cap expression. PK-15 cells were transfected 4.0 μg of pcDNA3.0-PCV4-Cap plasmid. The cells were fixed at 24, 48, and 72 h post-transfection (hpt) and incubated with the antibodies corresponding PCV4 Cap, and DDX21 followed by the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (green) and Alexa Fluor-546 conjugated donkey anti-rabbit IgG (red) secondary antibodies. Nuclei were stained with DAPI (blue) and then observed under a confocal microscope. Scale bar, 10μm. (B) The proportion of co-localization of PCV4 Cap and DDX21 proteins was analyzed using ImageJ software at 24, 48, and 72 hpt. (C) The three-layer dimensional confocal stack analysis was performed to verify the co-localization of PCV4 Cap and DDX21 proteins at 48 and 72 hpt. (D) The cell nuclear and cytoplasmic fractions were extracted after PK-15 cells transfected 4.0 μg of pcDNA3.0-PCV4-Cap plasmid. At 48 and 72 hpt, the protein samples were prepared and analyzed by western blotting using antibodies against PCV4 Cap and DDX21. Histone H3 and…

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  FIGURE 2. PCV4 Cap binds directly to DDX21. (A) PK-15 cells were transfected with empty vector or Flag-PCV4-Cap plasmid for 48 h. (B,C) The cell lysate extracts were immunoprecipitated with Flag beads (A,B) or anti-Myc purified IgG (C). (D) The His-sumo-PCV4 Cap mixed with the GST, GST-DDX21 proteins were GST pulled-down and then analyzed by western blotting using corresponding antibodies.

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  FIGURE 3. The N-terminal residues 1–37 of PCV4 Cap are essential for binding to DDX21. (A–C) HEK293T cells were co-transfected with plasmids containing full-length PCV4 Cap or truncated mutants fused with a GFP-, or Flag-GST tag, along with Flag-DDX21 plasmid for 48 h; the cell lysate extracts were immunoprecipitated with Flag beads (A) or anti-GFP purified IgG (B), or pulled-down with glutathione S-transferase (GST) beads (C) and then detected by western blotting using the indicated antibodies. (D) The nucleolar localization signals (NoLSs) within capsid protein of PCV1, 2, 3, 4 were responsible for the binding to DDX21. HEK293T cells were cotransfected with plasmids encoding NoLSs of PCV1, 2, 3, 4, along with Flag-DDX21; cell lysates were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. (E) Amino acid sequences alignment of the NoLSs from different PCV4 strains.

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  FIGURE 4. 763GSRSNRFQNK772 of DDX21 is crucial for binding to PCV4 Cap. (A) Schematic representation of the NTD, Helicase D, and CTD of DDX21 and their truncation mutants used in this study. (B–D) The DDX21-CTD-(582–784 aa) interacted with PCV4 Cap. HEK293T cells were co-transfected with expression plasmids GFP- DDX21-WT or its serial GFP-DDX21 truncated mutants M1 to M6, together with Flag-PCV4-Cap or Flag-gst-PCV4-Cap plasmid. The cell lysate extracts were immunoprecipitated or GST pulled-down followed by western blotting using the indicated antibodies. (E,F) Identification the critical amino acids of DDX21-CTD essential for interaction with PCV4 Cap. HEK293T cells were co-transfected with DDX21 or DDX21 truncated mutants M7 to M9, along with Flag-PCV4-Cap or Flag-gst-PCV4-Cap plasmid, and the cell lysate extracts were immunoprecipitated or GST pulled-down followed by western blotting using the indicated antibodies.

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  FIGURE 5. DDX21 knockdown abrogated the nucleolar localization of PCV4 Cap. (A) Immunofluorescence analysis of DDX21 protein in shCON-transfected and DDX21-silenced PK-15 cells. The cells were incubated with the DDX21 antibody followed by the Alexa Fluor-546 conjugated donkey anti-rabbit IgG (red) secondary antibody. Nuclei were stained with DAPI (blue) and then observed under a confocal microscope. Scale bar, 20 μm. (B) The fluorescence intensity of DDX21 was analyzed using ImageJ software in shCON- and DDX21-silenced PK-15 cells. (C) The knockdown efficiency of DDX21 in shCON-transfected and DDX21-silenced PK-15 cells. The cell lysates were subjected to immunoblotting using the indicated antibodies. (D) The viability of PK-15 cells stably expressing a short hairpin RNA (shDDX21) was analyzed with a cell counting kit-8 (CCK-8) assay. (E) DDX21-knocked down and shCON-transfected and DDX21-overexpressed PK-15 cells were, respectively, transfected with GFP-PCV4-Cap for 24 h, and the cells were fixed and incubated with the DDX21 antibody and then subjected to confocal microscopic observation. Nuclei were stained with DAPI. Scale bar, 10 μm. (F) The fluorescence intensity of PCV4 Cap in the…