Y-box-binding protein 1 stabilizes the capsid protein of porcine circovirus type 2 to promote viral replication

Figures

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  Fig. 1. YBX1 promotes the replication of PCV2. A Western blotting of YBX1 protein expression levels in PK-15 cells transfected with shYBX1 (YBX1-targeting shRNA). B Cell viability assessment of PK-15 cells stably expressing shYBX1, performed using the Cell Counting Kit 8 (CCK-8) assay. C and G Immunoblotting of Cap, Rep, YBX1, GFP, and β-actin proteins in either YBX1-silenced PK-15 cells (C) or PK-15 cells overexpressing YBX1 via transfection with the pWPXL-YBX1 plasmid (G). Cells were infected with PCV2 at an MOI of 1.0 and harvested at the indicated time points. PK-15 cells transfected with shCON (control shRNA) or empty pWPXL vector served as negative controls. F and J TCID50 values of PCV2 in samples derived from panels (C) and (G), respectively. YBX1-silenced or pWPXL-YBX1-overexpressing PK-15 cells were infected with PCV2 at an MOI of 1.0. Viral stocks were harvested at 12-h intervals from 24 to 72 h post-infection (hpi) (for F) or 48 to 72 hpi (for J). (D, E, H, I) The protein intensity band ratios of Cap and Rep were normalized to control conditions (β-actin) in panels (C, G), respectively. Data are presented as the mean ± SD from three independent biological experiments.…

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  Fig. 2. PCV2 Cap interacts directly with YBX1. A Co-localization of PCV2 Cap protein and YBX1. PK-15 cells were infected with PCV2 at an MOI of 1.0 and incubated for 48 or 72 h. Cells were fixed and then stained with mouse anti-Cap mAbs, rabbit anti-YBX1 antibodies, FITC-labeled goat anti-rabbit IgG (secondary antibody for YBX1 detection), and Alexa Fluor 546-conjugated donkey anti-mouse IgG (secondary antibody for Cap detection). Stained cells were visualized using a confocal laser scanning microscope. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). B Immunoprecipitation analysis of the endogenous Cap-YBX1 interaction. PK-15 cells were infected with PCV2 at an MOI of 1.0 for 48 h. Cell lysates were prepared and immunoprecipitated with purified anti-Cap mAbs, followed by immunoblotting with anti-Cap, anti-YBX1, and anti-β-actin antibodies. C PK-15 cells were transfected with either an empty vector (negative control) or a FLAG-Cap-expressing vector, cultured for 48 h, and then subjected to immunoprecipitation analysis. D and E Co-immunoprecipitation analysis of the exogenous Cap-YBX1 interaction. HEK293T cells were co-transfected with Myc-tagged Cap (Myc-Cap)…

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  Fig. 3. The 189RRRR192 residues of YBX1 bind to PCV2 Cap and regulate PCV2 replication. A Schematic diagram of YBX1 truncation mutants used in this study. B and C Co-immunoprecipitation (Co-IP) analysis confirming that the C-terminal domain (CTD) of YBX1 (amino acids [aa] 129 to 324) interacts with PCV2 Cap. HEK293T cells were co-transfected with plasmids encoding GFP-tagged full-length YBX1 (GFP-YBX1-WT) or a series of GFP-tagged YBX1 truncation mutants (M1-M6), together with a FLAG-Cap-expressing plasmid. Cell lysates were immunoprecipitated using FLAG beads (B) or purified anti-GFP IgG (C), followed by immunoblotting with the indicated antibodies. D Co-IP analysis identifying that aa 129–205 within the YBX1-CTD mediates interaction with PCV2 Cap. HEK293T cells were co-transfected with plasmids encoding GFP-YBX1-WT or GFP-YBX1 truncation mutants (M3, M7, M8), along with the FLAG-Cap plasmid. Cell lysates were subjected to immunoprecipitation with FLAG beads and immunoblotted using the indicated antibodies. E Schematic diagram of YBX1's D1–D6 truncation constructs used in this study. F and G Mapping of key amino acid residues within the YBX1-CTD required for Cap binding. HEK293T cells…

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  Fig. 4. YBX1 targets the NLS domain of PCV2 Cap to inhibit its degradation. A Schematic representation of PCV2 Cap truncation mutants used in this study. B and C Co-immunoprecipitation (Co-IP) analysis confirming interaction between the N-terminal NLS of PCV2 Cap and YBX1. HEK293T cells were co-transfected with a FLAG-YBX1 expression plasmid and plasmids encoding GFP-tagged full-length Cap, Cap lacking the NLS (Cap-ΔNLS), or GFP-tagged Cap-NLS truncation mutants. Cell lysates were immunoprecipitated using FLAG beads (B) or purified anti-GFP IgG (C), followed by immunoblotting with the indicated antibodies. The 'anti-FLAG' gel images between Input and IP: FLAG mAb may appear similar, although they were not duplicate (B). D Expression analysis of GFP-tagged Cap, Cap-ΔNLS, and isolated NLS in YBX1-deficient cells. YBX1-deficient cells were transfected with plasmids encoding GFP-Cap, GFP-Cap-ΔNLS, or GFP-NLS, and cultured for 48 h before protein detection. E Effect of YBX1 knockdown and proteasome inhibition on the expression of GFP-tagged Cap-NLS. shYBX1-or shCON-transduced PK-15 cells were transfected with the GFP-Cap-NLS plasmid; at 30 h post-transfection, cells were treated with DMSO…

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  Fig. 5. YBX1 knockdown facilitates PCV2 Cap degradation through the proteasome pathway. A Immunoblot analysis of Cap, YBX1, and β-actin proteins in YBX1-silenced PK-15 cells and control PK-15 cells. Cells were infected with PCV2 at an MOI of 10.0 or 20.0 and harvested at 21 or 24 hpi. β-actin served as a loading control. B Quantitative real-time PCR analysis of the relative Cap mRNA transcript levels in PCV2-infected cells (MOI = 10.0) at 21 and 24 h hpi. Data were analyzed using the 2−ΔΔCT method, with results normalized to a reference gene and presented relative to the control group. C Effect of cycloheximide (CHX) treatment on Cap protein stability in YBX1-silenced and control cells. Cells were first transfected with the pcDNA3.0-Cap plasmid for 36 h, then treated with CHX (100 μg/mL) for the indicated time periods. The Cap protein levels were detected by western blotting (β-actin as loading control). D Effect of the proteasome inhibitor MG132 or lysosome inhibitor chloroquine (CQ) treatment on Cap protein expression in shYBX1-or shCON-transduced PK-15 cells. Cells were transfected with pcDNA3.0-Cap; at 30 h post-transfection, they were treated with either DMSO (solvent control),…