DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication

Figures

• 

  FIGURE 1. DDX21 relocates from the nucleolus to the cytoplasm induced by PCV2 infection. (A) Immunofluorescence analysis of the subcellular localization of DDX21 during PCV2 infection. PK-15 cells were infected with PCV2 at a MOI of 1.0 for 24, 48, 72 h post-infection (hpi). Cells were fixed and stained with mouse mAb against Cap, rabbit anti-DDX21 antibody, FITC-labeled goat anti-mouse IgG, and Alexa Fluor 546-conjugated donkey anti-rabbit IgG. PK-15 cells were observed under a confocal microscope (Supplementary Figure 1). Nuclei were stained with DAPI. Scale bar, 10 μm. (B) The proportion of co-localization of PCV2 Cap and DDX21 proteins was analyzed using ImageJ software at 24, 48, and 72 hpi. (C) The nuclear and cytoplasmic fractions were extracted after PK-15 cells infected with PCV2 at a MOI of 1.0. At 48 and 72 hpi, the protein samples were prepared and analyzed by western blotting using antibodies against PCV2 Cap and DDX21. Histone H3 and β-tubulin served as fractionation quality controls. (D) The DDX21 protein band intensity was analyzed using ImageJ software at 48, 72 hpi. Data are presented as means ± SD of three independent experiments. *p < 0.05.

• 

  FIGURE 2. Cap interacts directly with DDX21. (A) Immunoprecipitation analysis. PK-15 cells were infected with PCV2 at a MOI of 1.0 for 48 h and cell lysates were immunoprecipitated with purified anti-Cap IgG mAb, followed by immunoblotting with anti-Cap, anti-DDX21, and anti-β-actin antibodies. Besides, the PCV2-infected PK-15 cell lysates were further immunoprecipitated with purified anti-Rep IgG mAb, followed by immunoblotting with anti-Rep, anti-DDX21, and anti-β-actin antibodies (Supplementary Figure 2). (B) PK-15 cells were transfected with an empty vector and FLAG-Cap for 48 h. (C,D) Co-immunoprecipitation analysis. HEK293T cells were co-transfected with Myc-Cap and FLAG-DDX21 for 48 h, and then the cell lysates were immunoprecipitated with Flag beads (B,C) or anti-Myc-purified IgG (D). (E) GST pull-down assays. GST or GST-DDX21 proteins were immobilized on GST beads and incubated with His-sumo-Cap. GST or GST-DDX21 proteins in the GST pull-down assays were examined using immunoblotting with anti-His, and anti-GST antibodies, respectively. GST, glutathione-S-transferase.

• 

  FIGURE 3. Binding domain identification of Cap with DDX21. HEK293T cells were co-transfected with plasmids containing full-length PCV2 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,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 binding to DDX21. 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-DDX21; cell lysates were subjected to immunoprecipitation and immunoblotting using the indicated antibodies.

• 

  FIGURE 4. 763GSRSNRFQNK772 of DDX21 is crucial for binding to 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-(582-784aa) interacted with 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-Cap or FLAG-GST-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 Cap. HEK293T cells were co-transfected with DDX21-WT or DDX21 truncated mutants M3, M7 to M9, along with FLAG-Cap plasmid, and the cell lysate extracts were immunoprecipitated followed by western blotting using the indicated antibodies.

• 

  FIGURE 5. DDX21 promotes the replication of PCV2. (A,C) Immunoblotting of the proteins Cap, Rep, DDX21, FLAG, and β-actin in DDX21-silenced or DDX21-overexpressing PK-15 cells. The cells were infected with PCV2 at a MOI of 1.0, and shCON-transfected or empty vector-transfected cells served as negative controls. (B,D) TCID50 values of PCV2 in samples from (A,C). Viral stocks were harvested at 48, 60, 72 hpi. Data are presented as mean ± SD from three independent biological experiments. ns, not significant; *p < 0.05.