Journal: Journal of Virology

Article Title: Chicken infectious anemia virus exploits host CK2α as an essential factor for its replication via VP2 Ser182/Asp183-mediated interaction

doi: 10.1128/jvi.01739-25

Figure Lengend Snippet: CIAV VP2 interacts with the cellular protein CK2α. ( A ) Pull-down analysis of VP2-interacting proteins in CIAV-infected MDCC-MSB1 cells. MDCC-MSB1 cells were either mock-infected or infected with CIAV. Cell lysates were subjected to pull-down using an anti-VP2 monoclonal antibody, and proteins were separated by SDS-PAGE. The protein band indicated by the arrow was excised and analyzed by LC-MS/MS to identify VP2-interacting partners. ( B ) Direct interaction between VP2 and CK2α. The recombinant glutathione S-transferase (GST)-CK2α protein was bound to anti-GST agarose beads at 4°C for 2 h, with the GST protein used as a control. The beads were incubated with His-tagged VP2 overnight at 4°C, after which the samples were eluted and analyzed by Western blot with anti-His and anti-GST antibodies. ( C and D ) Validation of the interaction between VP2 and endogenous CK2α in CIAV-infected cells. MDCC-MSB1 cells were mock-infected or infected with CIAV at a multiplicity of infection (MOI) of 1. At 48 h post-infection, co-immunoprecipitation (co-IP) assays were performed using ( C ) an anti-VP2 monoclonal antibody or an ( D ) anti-CK2α monoclonal antibody. Immune complexes were analyzed by Western blot: ( C ) CK2α was detected using an anti-CK2α antibody, CK2α′ was detected using an anti-CK2α′ antibody; ( D ) VP2 was detected using an anti-VP2 antibody. “Isotype” denotes the IgG1 isotype control antibody for the respective primary antibody (anti-VP2 or anti-CK2α). ( E ) Colocalization of CIAV VP2 and endogenous CK2α in CIAV-infected cells. MDCC-MSB1 cells were mock-infected or infected with CIAV (MOI = 1). At 24 h post-infection, cells were fixed, permeabilized, and stained with a mouse anti-VP2 antibody and a rabbit anti-CK2α antibody. Secondary antibodies used were fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (green, for VP2) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (red, for CK2α). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Images were acquired using a confocal laser scanning microscope. Scale bar = 10 µm. Colocalization was assessed by calculating the Pearson correlation coefficient with ImageJ/Fiji software.

Article Snippet: Anti-CK2α (CSNK2A1; A19683) antibody was obtained from ABclonal Technology (Wuhan, China).

Techniques: Infection, SDS Page, Liquid Chromatography with Mass Spectroscopy, Recombinant, Control, Incubation, Western Blot, Biomarker Discovery, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining, Laser-Scanning Microscopy, Software

Journal: Journal of Virology

Article Title: Chicken infectious anemia virus exploits host CK2α as an essential factor for its replication via VP2 Ser182/Asp183-mediated interaction

doi: 10.1128/jvi.01739-25

Figure Lengend Snippet: CIAV infection upregulates CK2α expression in chicken thymus tissues. ( A, B ) CK2α expression in CIAV-infected MDCC-MSB1 cells. ( A ) Time-course analysis: MDCC-MSB1 cells were mock-infected or infected with CIAV (MOI = 1), then harvested at 12, 24, 48, and 72 hpi. Western blot analysis was performed using anti-VP2 (viral infection marker), anti-CK2α, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control) antibodies. ( B ) Dose-response analysis: MDCC-MSB1 cells were mock-infected or infected with CIAV at the indicated MOIs, then harvested at 24 hpi. Western blot analysis was conducted as described in panel A . ( C, D ) Viral VP2 and host CK2α mRNA expression in tissues of CIAV-infected chickens. Mock-infected ( n = 3) or CIAV-infected ( n = 3) chickens were euthanized, and tissues from different organs were collected. Total RNA was extracted, and ( C ) VP2 (viral load marker) and ( D ) CK2α mRNA levels were quantified by quantitative PCR (qPCR). GAPDH served as an internal control. Relative mRNA levels were calculated as the ratio of target gene (VP2/CK2α) mRNA to GAPDH mRNA in the same sample. ( C ) VP2 expression was normalized to the VP2 level in the thymus of CIAV-infected chickens (set to 1.0). ( D ) CK2α expression was normalized to the CK2α level in the thymus of mock-infected chickens (set to 1.0). The y -axis is presented on a base-10 logarithmic scale to visualize large differences in expression. Data are representative of three independent experiments and presented as means ± standard deviation (SD). Statistical significance: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Article Snippet: Anti-CK2α (CSNK2A1; A19683) antibody was obtained from ABclonal Technology (Wuhan, China).

Techniques: Infection, Expressing, Western Blot, Marker, Control, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Virology

Article Title: Chicken infectious anemia virus exploits host CK2α as an essential factor for its replication via VP2 Ser182/Asp183-mediated interaction

doi: 10.1128/jvi.01739-25

Figure Lengend Snippet: CIAV replication requires CK2α. ( A ) Efficacy of CK2α knockdown by RNAi. MDCC-MSB1 cells were transfected with CK2α-specific siRNA or control siRNA (double transfections at 24 h intervals). At 24 h after the second transfection, cell lysates were analyzed by Western blot using an anti-CK2α antibody. Tubulin served as a loading control. ( B ) Densitometric quantification of CK2α band intensity in panel A , normalized to GAPDH. ( C, D ) Effect of CK2α knockdown on CIAV VP2 expression. MDCC-MSB1 cells were transfected with CK2α siRNA or control siRNA, then mock-infected or infected with CIAV (MOI = 1). ( C ) At 24 and 48 hpi, Western blot analysis was performed using anti-VP2, anti-CK2α, and anti-GAPDH antibodies (GAPDH as loading control). ( D ) Densitometric quantification of VP2 bands in panel C , expressed as the ratio of VP2 levels in siRNA-transfected cells to those in untreated cells. ( E ) Impact of CK2α knockdown on CIAV growth kinetics. MDCC-MSB1 cells were transfected with CK2α siRNA or control siRNA, then infected with CIAV (MOI = 1). Viral genome copies were quantified by qPCR at 12, 24, 48, and 72 hpi. (F–H) Rescue experiments with wild-type and kinase-inactive CK2α. MDCC-MSB1 cells were transfected with CK2α siRNA (or control siRNA), then transfected with pEGFP-N1-CK2α (wild-type), pEGFP-N1-CK2αK68M (kinase-inactive mutant), or pEGFP-N1 (empty vector) prior to CIAV infection (MOI = 1). ( F ) Western blot analysis at 48 hpi using anti-VP2, anti-CK2α, anti-green fluorescent protein (GFP), and anti-GAPDH antibodies. ( G ) Densitometric quantification of VP2 bands in panel F , expressed as the ratio of VP2 levels in transfected cells to those in untreated cells. ( H ) Viral genome copies quantified by qPCR at 72 hpi. ( I ) Cytotoxicity of the CK2α inhibitor CX-4945. MDCC-MSB1 cell viability was assessed by CCK-8 assay 24 h after treatment with CX-4945. ( J ) Effect of CX-4945 on CIAV growth kinetics. MDCC-MSB1 cells were infected with CIAV (MOI = 1), treated with 10 μM CX-4945 2 h post-infection, and viral genome copies were quantified by qPCR at 12, 24, 48, and 72 hpi. ( K, L ) Dose-dependent effect of CX-4945 on VP2 expression. MDCC-MSB1 cells were mock-infected or infected with CIAV (MOI = 1), then treated with CX-4945 (0.1, 1, 10 μM) or dimethyl sulfoxide (DMSO) (control) 2 h post-infection. ( K ) Western blot analysis at 24 hpi using anti-CK2α, anti-VP2, and anti-GAPDH antibodies. ( L ) Densitometric quantification of VP2 bands in panel K , expressed as the ratio of VP2 to GAPDH band densities. Data are representative of three independent experiments and presented as means ± SD. Statistical significance: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Article Snippet: Anti-CK2α (CSNK2A1; A19683) antibody was obtained from ABclonal Technology (Wuhan, China).

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Infection, Mutagenesis, Plasmid Preparation, CCK-8 Assay

Journal: Journal of Virology

Article Title: Chicken infectious anemia virus exploits host CK2α as an essential factor for its replication via VP2 Ser182/Asp183-mediated interaction

doi: 10.1128/jvi.01739-25

Figure Lengend Snippet: CK2α kinase stabilizes VP2 by inhibiting its proteasomal degradation. (A–C) Effect of CK2α knockdown on CIAV viral gene transcription. MDCC-MSB1 cells were transfected with CK2α-specific siRNA or control siRNA, then infected with CIAV (MOI = 1). At 6, 12, and 24 hpi, qPCR was performed to quantify the mRNA expression levels of VP1 ( A ), VP2 ( B ), and VP3 ( C ). GAPDH served as an internal control. Relative gene expression was calculated as the ratio of viral gene mRNA to GAPDH mRNA at each time point. ( D, E ) VP2 stability under CK2α knockdown. MDCC-MSB1 cells were transfected with CK2α siRNA (or control siRNA) and co-transfected with pRK5-flag-VP2. Twelve hours post-transfection, protein synthesis was inhibited by adding cycloheximide (CHX, 50 µg/mL), and cells were harvested at 0, 6, 12, and 24 h post-CHX treatment. ( D ) Western blot analysis of cell lysates using anti-Flag (for Flag-VP2), anti-CK2α, and anti-Tubulin (loading control) antibodies. ( E ) Densitometric quantification of Flag-VP2 bands in panel D , normalized to Tubulin. The normalized Flag-VP2 level at 0 h post-CHX was set to 1.0. ( F, G ) VP2 stability under CK2α kinase inhibition. MDCC-MSB1 cells were pre-treated with 10 μM CX-4945 (CK2α inhibitor) or DMSO (solvent control), then transfected with pRK5-flag-VP2. Twelve hours post-transfection, cells were treated with CHX (50 µg/mL) and harvested at 0, 6, 12, and 24 h post-CHX. ( F ) Western blot analysis using anti-Flag, anti-CK2α, and anti-Tubulin antibodies. ( G ) Densitometric quantification of Flag-VP2 bands in panel F , normalized to Tubulin. The normalized Flag-VP2 level at 0 h post-CHX was set to 1.0. ( H ) Effect of degradation pathway inhibitors on VP2 stability during CK2α knockdown. MDCC-MSB1 cells were transfected with CK2α siRNA (or control siRNA) and pRK5-Flag-VP2. Twelve hours post-transfection, cells were treated for 12 h under four conditions: (i) untreated (baseline); (ii) CHX (50 µg/mL) + 0.1% DMSO (protein synthesis inhibition); (iii) CHX + 20 µM MG132 (proteasome inhibition); (iv) CHX + 20 µM chloroquine (lysosome inhibition). Cell lysates were analyzed by Western blot using anti-Flag, anti-CK2α, and anti-Tubulin antibodies. ( I ) Effect of degradation pathway inhibitors on VP2 stability during CK2α inhibition. MDCC-MSB1 cells were pre-treated with DMSO or CX-4945, then transfected with pRK5-Flag-VP2. Twelve hours post-transfection, cells were treated under the same four conditions as in panel H . Western blot analysis was performed using anti-Flag, anti-CK2α, and anti-Tubulin antibodies. Data are representative of three independent experiments and presented as means ± SD. Statistical significance: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Article Snippet: Anti-CK2α (CSNK2A1; A19683) antibody was obtained from ABclonal Technology (Wuhan, China).

Techniques: Knockdown, Transfection, Control, Infection, Expressing, Gene Expression, Western Blot, Inhibition, Solvent

Journal: Journal of Virology

Article Title: Chicken infectious anemia virus exploits host CK2α as an essential factor for its replication via VP2 Ser182/Asp183-mediated interaction

doi: 10.1128/jvi.01739-25

Figure Lengend Snippet: Ser182 and Asp183 of CIAV VP2 are critical for VP2-CK2α interaction. ( A ) Schematic representation of the full-length CIAV VP2 protein (amino acid [aa] positions indicated). ( B ) Schematic diagrams of the genes encoding full-length VP2 and its truncated mutants (Δ1 to Δ5). The amino acid ranges of each truncation are labeled to indicate deleted regions relative to full-length VP2. ( C–H ) Mapping of the CK2α-binding domain in VP2 using truncated mutants. DF-1 cells were transfected with pEGFP-N1-VP2 (full-length VP2), pEGFP-N1-VP2 truncated mutants, or empty pEGFP-N1 vector (negative control). At 24 h post-transfection, cell lysates were subjected to immunoprecipitation (IP) using an anti-GFP monoclonal antibody (to pull down EGFP-tagged VP2/fragments). IP pellets were analyzed by Western blot with: (i) anti-GFP antibody (to detect EGFP-VP2/fragments), (ii) anti-CK2α antibody (to detect co-immunoprecipitated CK2α), and (iii) anti-Tubulin or anti-GAPDH antibody (loading control for input lysates). Input lysates (prior to IP) were also probed to confirm expression of EGFP-VP2/fragments and endogenous CK2α.（ C ) VP2 truncation mutants Δ1–Δ5.( D, E ) VP2 single-point mutations within aa 172–187. ( F ) VP2 single-point mutations S182A and D183A, and the double mutation S182A/D183A.(G)VP2 K102D mutation.(H) VP2 double mutation S182A/D183A.Ser182 and Asp183 of CIAV VP2 are critical for VP2-CK2α interaction.

Article Snippet: Anti-CK2α (CSNK2A1; A19683) antibody was obtained from ABclonal Technology (Wuhan, China).

Techniques: Labeling, Binding Assay, Transfection, Plasmid Preparation, Negative Control, Immunoprecipitation, Western Blot, Control, Expressing, Mutagenesis

Journal: Journal of Virology

Article Title: Chicken infectious anemia virus exploits host CK2α as an essential factor for its replication via VP2 Ser182/Asp183-mediated interaction

doi: 10.1128/jvi.01739-25

Figure Lengend Snippet: Double mutations S182A and D183A of VP2 impaired CIAV replication. ( A ) Schematic diagram of CIAV VP2 highlighting the amino acid mutation sites: Ser182 (S182) and Asp183 (D183) (mutated to alanine in the VP2 mutant, denoted as “mt”). ( B ) Effect of VP2 mutations on interaction with endogenous CK2α. MDCC-MSB1 cells were mock-infected or infected with CIAV-WT, CIAV-VP2/mt, or CIAV-Revertant (Rev) (MOI = 1). At 48 hpi, co-IP was performed using an anti-VP2 mAb. The quantity of antibody-conjugated beads was optimized to ensure saturation of binding sites, allowing for comparable immunoprecipitation efficiency across samples. Co-immunoprecipitated CK2α was detected by Western blot using an anti-CK2α antibody. “Isotype” refers to the IgG1 isotype control antibody for the anti-VP2 mAb. Input lysates were probed to confirm expression of VP2 and CK2α. (C–E) Viral protein expression in cells infected with CIAV-VP2/mt. MDCC-MSB1 cells were infected with CIAV-WT, CIAV-VP2/mt, or CIAV-Rev (MOI = 1). ( C ) At 24 and 48 hpi, Western blot analysis of cell lysates was performed using anti-VP1, anti-VP2, and anti-GAPDH (loading control) antibodies. Densitometric quantification of ( D ) VP1 and ( E ) VP2 band intensities in panel C , normalized to GAPDH. ( F ) Growth kinetics of CIAV VP2/mt. MDCC-MSB1 cells were infected with CIAV-WT, CIAV-VP2/mt, or CIAV-Rev (MOI = 1). Viral genome copies were quantified by qPCR at 12, 24, 48, and 72 hpi. ( G ) Genetic stability of VP2-targeted mutations. CIAV-VP2/mt was serially passaged 10 times in MDCC-MSB1 cells. Viral genomic DNA was extracted, and the VP2 mutation region was amplified by PCR. Ten single clones were subjected to Sanger sequencing to verify the stability of S182/D183 mutations. ( H, I ) VP2 stability in cells infected with CIAV VP2/mt. MDCC-MSB1 cells were infected with CIAV-WT, CIAV-VP2/mt, or CIAV-Rev. At 24 hpi, protein synthesis was inhibited by CHX (50 µg/mL), and cells were harvested at 0, 6, 12, and 24 h post-CHX treatment. ( H ) Western blot analysis of cell lysates using anti-VP2 and anti-Tubulin (loading control) antibodies. ( I ) Densitometric quantification of VP2 band intensities in panel H , normalized to Tubulin. The normalized VP2 level at 0 h post-CHX was set to 1.0. ( J, K ) VP2 stability of the S182A phospho-deficient mutant. MDCC-MSB1 cells were transfected with pRK5-Flag-VP2 or pRK5-Flag-VP2(S182A). Twelve hours post-transfection, protein synthesis was inhibited by CHX (50 µg/mL), and cells were harvested at 0, 6, 12, and 24 h post-CHX treatment. ( J ) Western blot analysis of cell lysates using anti-Flag, anti-CK2α, and anti-Tubulin (loading control) antibodies. ( K ) Densitometric quantification of Flag-VP2 band intensities in panel J , normalized to Tubulin. The normalized level of each protein at 0 h post-CHX was set to 1.0. Data are representative of three independent experiments and presented as means ± SD. Statistical significance: ***, P < 0.001; **, P < 0.01; *, P < 0.05. “mt” = CIAV-VP2/mutant; “Rev” = CIAV-Revertant.

Article Snippet: Anti-CK2α (CSNK2A1; A19683) antibody was obtained from ABclonal Technology (Wuhan, China).

Techniques: Mutagenesis, Infection, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Western Blot, Control, Expressing, Amplification, Clone Assay, Sequencing, Transfection