DDX21 Promotes PCV3 Replication by Binding to Cap Protein and Inhibiting Interferon Responses

Figures

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  Figure 1. PCV3 infection prevents the repression of DDX21 at protein and mRNA levels. (A) PK-15 cells were inoculated with PCV3 at a MOI of 1.0 or mock-infected for 6, 12, 24, and 36 h and analyzed by immunoblotting with anti-DDX21, anti-Rep, and anti-β-actin antibodies, respectively. (B) qRT-PCR measurements of the DDX21 mRNA at 6, 12, 24, and 36 hpi. (C,D) Proteins and DDX21 mRNA were extracted from PCV3-inoculated or mock-infected 3D4/21 cells at 6, 12, 24, and 36 h and analyzed by immunoblotting (C) and RT-qPCR (D), respectively. (E,G) PK-15 and 3D4/21 cells were inoculated with PCV3 at the indicated MOI of 0.2, 1.0, 5.0 or mock-infected for 24 h, and analyzed by immunoblotting respectively. (F,H) RT-qPCR measurements of the DDX21 mRNA in PK-15 or 3D4/21 cells infected with PCV3 at the indicated MOI of 0.2, 1.0, 5.0 or mock-infected for 24 h. * p < 0.05.

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  Figure 2. DDX21 expression facilitates PCV3 replication. (A,C) DDX21-overexpressing or DDX21-silenced PK-15 cells were inoculated with PCV3 at a MOI of 1.0 for 24, 36 hpi, and immunoblotting of Rep, DDX21, FLAG, and β-actin proteins. (B,D) TCID50 values of PCV3 in samples from (A,C). Viral stocks were harvested at 24, 36 hpi, and shCON-transfected or empty vector-transfected cells served as negative controls. Data are presented as mean ± SD from three independent biological experiments. * p < 0.05.

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  Figure 3. PCV3 Cap colocalizes and directly interacts with DDX21. (A) The co-localization of the viral protein Cap with DDX21. PK-15 cells were co-transfected with GFP-DDX21 and mCherry-PCV3-Cap for 24 h. (B,C) The co-localization of the viral protein DDX21 with PCV3 Cap or NCL. HEK293T cells were co-transfected with GFP-DDX21 and mCherry-PCV3-Cap (B) or mCherry-NCL (C) for 24 h. Cells were stained with DAPI and then observed under a confocal microscope. (D) Immunoprecipitation analysis. PK-15 cells were transfected with FLAG-PCV3-Cap for 48 h. (E,F) HEK293T cells were co-transfected with FLAG-PCV3-Cap and GFP-DDX21 or FLAG-DDX21 and GFP-PCV3-Cap for 48 h, and the cell lysates were immunoprecipitated with FLAG beads and then analyzed by immunoblotting with anti-FLAG, anti-GFP, and anti-β-actin antibodies. (G) The PCV1, 2, 3, 4 Cap proteins were indispensable to interaction with DDX21. (H) Sumo-PCV3-Cap bound to DDX21. GST or GST-DDX21 proteins were immobilized on GST beads and incubated with His-Sumo-PCV3-Cap. The targeted proteins were examined using immunoblotting with anti-His and anti-GST antibodies, respectively.

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  Figure 4. The PCV3 Cap NLS is crucial for binding to DDX21. (A) Schematic representation of the truncation mutants of PCV3 Cap used in this study. (B–D) The PCV3 Cap NLS interacted with DDX21. HEK293T cells were co-transfected with GFP-PCV3-Cap-WT, -PCV3-Cap-M1, -PCV3-Cap-M2 or FLAG-GST-PCV3-Cap-WT, -PCV3-Cap-M1, -PCV3-Cap-M2 with FLAG-DDX21, and cell lysates were then subjected to immunoprecipitation or GST pull-down assays. (E,F) HEK293T cells were co-transfected with Cap-NLSs within PCV1, 2, 3, 4 (E) and circoviruses from different species (F), along with FLAG-DDX21, and cell lysates were then subjected to immunoprecipitation assays.

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  Figure 5. The C-terminal domain of DDX21 mediated interaction with PCV3 Cap and facilitated viral replication. (A) Schematic representation of three functional domains of DDX21 used in this study. (B–E) The helicase domain of DDX21-(763-772aa) bound to PCV3 Cap. HEK293T cells were co-transfected with a serial of GFP-DDX21 variants M1 to M9 with FLAG-PCV3-Cap or FLAG-GST-PCV3-Cap, and cell lysates were then subjected to immunoprecipitation or GST pull-down assays. (F–H) PK-15 cells transfected with DDX21-WT, -M3, and -M4 for 24 h were inoculated with PCV3 at a MOI of 1.0 for 24, 36 hpi. Viral protein and titers were then determined by immunoblotting with anti-GFP, anti-Rep, and anti-β-actin antibodies and TCID50, respectively. * p < 0.05.

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  Figure 6. DDX21 inhibits IFN-β production and reduces ISG expression to promote PCV3 replication. (A–E) qRT-PCR was used to measure the relative IFN-β, MX1, MX2, OAS1, and ISG15 in DDX21-silenced 3D4/21 cells inoculated with PCV3 at a MOI of 1.0 for 24, 36 hpi. (F) Immunoblotting of the proteins p-TBK1, TBK1, p-IRF3, IRF3, Rep, FLAG, and β-actin in DDX21-silenced 3D4/21 cells inoculated with PCV3 for 24, 36 hpi. (G–I) qRT-PCR was used to measure the relative IFN-β, MX1, MX2, in DDX21-overexpressing 3D4/21 cells inoculated with PCV3 at a MOI of 1.0 for the indicated time. (J) Immunoblotting of the proteins p-TBK1, TBK1, p-IRF3, IRF3, Rep, DDX21, and β-actin in samples from (G–I). Data are presented as mean ± SD from three independent biological experiments. ns, not significant, p > 0.05; * p < 0.05.