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Phosphorylation of VP1 Mediated by CDK1-Cyclin B1 Facilitates Infectious Bursal Disease Virus Replication - PubMed

  • ️Sun Jan 01 2023

Phosphorylation of VP1 Mediated by CDK1-Cyclin B1 Facilitates Infectious Bursal Disease Virus Replication

Xifeng Hu et al. J Virol. 2023.

Abstract

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Avibirnavirus in the family Birnaviridae. It can cause serious failure of vaccination in young poultry birds with impaired immune systems. Post-translational modifications of the VP1 protein are essential for viral RNA transcription, genome replication, and viral multiplication. Little information is available so far regarding the exact mechanism of phosphorylation of IBDV VP1 and its significance in the viral life cycle. Here, we provide several lines of evidence that the cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex phosphorylates VP1, which facilitates viral replication. We show that the CDK1-cyclin B1 specifically interacts with VP1 and phosphorylates VP1 on the serine 7 residue, located in the N-terminal 7SPAQ10 region, which follows the optimal phosphorylation motif of CDK1, p-S/T-P. Additionally, IBDV infection drives the cytoplasmic accumulation of CDK1-cyclin B1, which co-localizes with VP1, supporting the kinase activity of CDK1-cyclin B1. Treatment with CDK1 inhibitor RO3306 and knockdown of CDK1-cyclin B1 severely disrupts the polymerase activity of VP1, resulting in diminished viral replication. Moreover, the replication of S7A mutant recombinant IBDV was significantly decreased compared to that of wild-type (WT) IBDV. Thus, CDK1-cyclin B1 is a crucial enzyme which phosphorylates IBDV VP1 on serine 7, which is necessary both for the polymerase activity of VP1 and for viral replication. IMPORTANCE Infectious bursal disease virus still poses a great economic threat to the global poultry farming industry. Detailed information on the steps of viral genome replication is essential for the development of antiviral therapeutics. Phosphorylation is a common post-translational modification in several viral proteins. There is a lack of information regarding the significance of VP1 phosphorylation and its role in modulating the viral life cycle. In this study, we found that CDK1-cyclin B1 accumulates in the cytoplasm and phosphorylates VP1 on serine 7. The presence of a CDK1 inhibitor and the silencing of CDK1-cyclin B1 decrease IBDV replication. The mutation of VP1 serine 7 to alanine reduces VP1 polymerase activity, disrupting the viral life cycle, which suggests that this residue serves an essential function. Our study offers novel insights into the regulatory mechanism of VP1 phosphorylation.

Keywords: CDK1-cyclin B1; IBDV VP1; Ser7; host kinase; phosphorylation; polymerase activity; viral polymerase; viral protein phosphorylation; virology; virus replication.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1

Cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex interacts with VP1 (A) Schematic diagram of the infectious bursal disease virus (IBDV) genome. (B) HEK-293T (293T) cells transiently transfected with Flag-VP1 (empty vectors as a negative control). Flag-VP1 was immunoprecipitated (IP) with anti-Flag agarose, separated by SDS-PAGE and stained with Coomassie brilliant blue. Separated specific bands were excised and analyzed by mass spectrometry. Four host kinases were associated with VP1, including cyclin-dependent kinase 1. (C) Coverage of identified peptides in the complete length of CDK1. (D) Identified peptides located in the CDK1 sequence. (E) Immunoblotting (IB) of whole-cell lysates (bottom) and proteins immunoprecipitated with anti-Flag agarose (top) from 293T cells transfected with the indicated plasmids for 36 h. (F) 293T cells were co-transfected with Flag-VP1- and Myc-cyclin B1-expressing plasmids for 36 h. Cells were then lysed and IP by anti-Flag agarose. The IP complex and whole-cell lysates were subjected to Western blotting using the indicated antibodies. (G) IB of whole-cell lysates (bottom) and proteins immunoprecipitated with anti-Flag agarose (top) from 293T cells co-transfected with Flag-VP1-, Myc-CDK1-, and Myc-cyclin B1-expressing plasmids for 36 h. (H) Purified GST-VP1 protein was separately incubated with purified His-CDK1 and His-cyclin B1 protein, followed by IP with glutathione S-transferase (GST) resin. IP complex was analyzed by Western blotting with indicated antibodies. The whole protein was examined by SDS-PAGE and visualized by Coomassie brilliant blue staining.

FIG 2
FIG 2

IBDV infection promotes expression of CDK1-cyclin B. (A) DF-1 cells were infected with IBDV at different times. Lysates of the infected DF-1 cells were subjected to Western blotting with the indicated antibodies. (B and C) Real-time quantitative PCR measurements of CDK1 (B) and cyclin B1 (C) mRNA abundances at the indicated times post-IBDV infection. Expression was normalized to the gapdh mRNA level. (D) DF-1 cells were infected with IBDV (MOI = 1) at the indicated times. Nuclear and cytoplasmic proteins were separated. CDK1, cyclin B1, CDK1Y15, VP1, β-actin and H3 protein level were analyzed by Western blotting. (E and F) Nuclear (E) and cytoplasmic (F) Western blot data were semi-quantified and normalized against β-actin and H3 protein loading control, respectively. (G) Confocal microscopy immunofluorescence images of mock-infected or IBDV (red)-infected DF-1 cells transfected with Myc-CDK1 (green) or Myc-cyclin B1 (green). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; blue). White arrows indicate VP1 puncta which co-localized with CDK1 or cyclin B1. Scale bars = 5 μm. (H and I) Quantified numbers of CDK1- (H) and cyclin B1-positive (I) cells in the nucleus and cytoplasm. Data were obtained from three independent experiments (n = 3) and are presented as means ± standard deviation (SD). ***, P < 0.001.

FIG 3
FIG 3

CDK1 inhibitor impairs IBDV replication. (A) DF-1 cells were treated with the indicated concentrations of RO3306 or dimethyl sulfoxide (DMSO). Treated cells were subjected to a cell viability assay as determined by a CCK8 kit. (B) DF-1 cells were treated with 5 μg/mL RO3306 or DMSO for 2, 6, 10, and 12 h. Whole-cell lysates were collected for Western blot analysis with the indicated antibodies. (C–D) DF-1 cells treated with 5 μg/mL RO3306 or DMSO for 6 h were infected with IBDV for an additional 6 and 12 h. Total viral RNA expression and major viral protein (VP1/2/3) expression were analyzed by reverse transcription-quantitative PCR (RT-qPCR) and Western blotting, respectively. (E) Comparison of VP1/2/3 expression in the different lanes in panel D. The densities of VP1/2/3 and β-actin were quantified by ImageJ software. (F) DF-1 cells treated with 5 μg/mL RO3306 or DMSO for 6 h were infected with IBDV for an additional 12, 18, and 24 h. Viral titers of whole cells were examined by 50% tissue culture infective dose (TCID50) assay. Western blot data were semi-quantified and normalized against the β-actin protein loading control. Data were obtained from three independent experiments and presented as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4
FIG 4

CDK1-cyclin B1 silencing inhibits viral proliferation. (A) Three small interfering RNAs (siRNAs) targeting CDK1, three cyclin B1 siRNAs, and the control siRNA (sictr) were individually transfected into DF-1 cells for 48 h. Whole-cell lysates were subjected to immunoblotting with corresponding antibodies (bottom panel). Comparison of CDK1 and cyclin B1 expression in different lanes. CDK1 and cyclin B1 density were quantified using ImageJ software (top panel). (B) Indicated siRNAs were co-transfected into DF-1 cells for 48 h. The protein levels of CDK1, cyclin B1 and β-actin in transfected cells were examined by Western blotting with indicated antibodies. (C and D) DF-1 cells transfected with indicated siRNAs for 48 h were infected with 1 MOI IBDV for different times. Viral genome (C) and major viral protein VP1/2/3 levels (D) were analyzed by RT-qPCR and Western blotting, respectively. (E) Comparison of VP1/2/3 expression in the different lanes in panel D. VP1/2/3 and β-actin density were quantified using ImageJ software. (F) DF-1 cells transfected with indicated siRNAs for 48 h were infected with 0.1 MOI IBDV for additional different times. Viral titers of whole cells were examined by TCID50 assay. Western blot data were semi-quantified and normalized against β-actin protein loading control. Data were obtained from three independent experiments and are presented as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 5
FIG 5

CDK1-cyclin B1 phosphorylates VP1. (A) Schematic diagram of published substrates and putative VP1 phosphorylation sites of CDK1. The multiple S/T-P motifs, positions of S7, S46, S342, S359, S563, and T811, are indicated. (B) 293T cells transfected with Flag-VP1 were treated with 10 nM nocodazole for different durations. Next, the cells were subjected to IP. Immunoblotting of IP complex (Top panel) and the cell lysates (Bottom panel) by Western blotting with indicated antibodies. (C) 293T cells transfected with Flag-VP1 were treated with 10 nM nocodazole or 10 nM nocodazole together with 5 μg/mL RO3306 for an additional 12 h. Next, cells were subjected to IP. Immunoblotting of IP complex (Top panel) and the cell lysates (Bottom panel) by Western blotting with indicated antibodies. (D) The indicated expressing plasmids were co-transfected into 293T cells for 36 h. Transfected cells were subjected to IP. The protein levels of IP complex and cell lysates were analyzed by Western blotting with recommended antibodies. (E) IB of whole-cell lysates (Bottom panel) and proteins immunoprecipitated with anti-Flag agarose (top panel) from 293T cells transfected with the indicated plasmids for 36 h.

FIG 6
FIG 6

Serine 7 of VP1 is the phosphorylation site. (A) 293T cells were transiently transfected with Flag-VP1- or Myc-CDK1AF-expressing plasmids for 36h. Flag-VP1 was IP with an anti-Flag agarose and separated by SDS-PAGE, and the band corresponding to VP1 (white box) was excised, digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS; left panel). The proteins in cell lysates were also analyzed by Western blotting. The MS spectra of the selected phosphorylated VP1 peptide, MSDIFNSPQAR, is shown in the right panel. (B) Flag-VP1-, Myc-CDK1-, and Myc-cyclin B1-expressing plasmids were co-transfected into 293T cells for 36 h. Flag-VP1 was purified by IP with an anti-Flag agarose and separated by SDS-PAGE, and the band corresponding to VP1 (white box) excised, digested, and analyzed by LC-MS (left panel). The proteins in cell lysates were also detected by Western blotting. The MS spectra of the selected phosphorylated VP1 peptide, MSDIFNSPQAR, is shown in the right panel. (C) Information on the phosphorylated peptide identified by LC-MS, as described in panels A and B. (D) Wild-type (WT) Flag-VP1 and six mutant VP1-expressing plasmids were individually co-transfected with Myc-CDK1AF into 293T cells for 36 h. IB of whole-cell lysates (Bottom panel) and proteins immunoprecipitated with anti-Flag agarose (top) from the transfected cells. (E) Quantitation shows mean p-S/T-P normalized to Flag-VP1 signals shown in panel D. p-S/T-P, Flag-VP1, and β-actin densities were quantified using ImageJ software. Data were obtained from three independent experiments and are presented as means ± SD. ***, P < 0.001.

FIG 7
FIG 7

Characterization of specific antibody against VP1 S7 phosphorylation. (A) Synthetic phosphopeptides and non-phosphopeptides were diluted and dotted on nitrocellulose membranes, then analyzed by dot blotting with S7 antibody. Peptide sequences are shown with the serine residue of interest indicated in red. (B) Flag-VP1 was co-transfected with indicated siRNAs into DF-1 cells for 36 h. The transfected cells were subjected to Western blotting with indicated antibodies. (C) 293T cells were transfected with the indicated expressing plasmids for 36 h. Cells were then subjected to IP by anti-Flag agarose. Immunoblotting of IP complex (top panel) and cell lysates (bottom) were performed to analyze indicated protein levels. (D) Purified His-VP1 or S7A proteins were incubated with purified His-CDK1/cyclin B1 kinases for phosphorylation reaction in vitro. The reaction complexes were analyzed by Western blotting with the indicated antibodies. The purified proteins were separately by SDS-PAGE and visualized by Coomassie brilliant blue staining. (E) DF-1 cells were infected with 1 MOI IBDV for different durations. Immunoblotting of cell lysates was performed to analyze the indicated proteins.

FIG 8
FIG 8

Phosphorylation of VP1 contributes to its polymerase activity. (A) The evaluation system for polymerase activity. (B) VP1- (or D402 and D416 mutant VP1), VP3-, and pUC-mA-expressing plasmids were transfected into 293T cells for 72 h. Cell lysates were subjected Western blotting to analyze VP2, VP1, VP3 and β-actin levels. (C) Top panel: 293T cells were transfected with indicated expressing plasmids for 72 h. Immunoblotting of cell lysates was performed by Western blotting with indicated antibodies. Bottom panel: comparison of VP2 expression in the different lanes shown in the top panel. VP2 and β-actin densities were quantified using ImageJ software. (D) Top panel: DF-1 cells were transfected with the indicated expressing plasmids for 72 h. Immunoblotting of cell lysates was performed by Western blotting with the indicated antibodies. Bottom panel: comparison of VP2 expression in the different lanes shown in the top panel. VP2 and β-actin densities were quantified using ImageJ software. (E) Top panel: DF-1 cells were transfected with the indicated expressing plasmids for 72 h. Transfected cells were treated with different concentrations of RO3306 (1, 3, and 5 μg) for 12 h before being harvested. Immunoblotting of cell lysates was performed by Western blotting with the indicated antibodies. Bottom panel: comparison of VP2 expression in the different lanes shown in the top panel. (F) Top panel: 293T cells were transfected with the indicated plasmids for 72 h. Immunoblotting of cell lysates was performed by Western blotting with the indicated antibodies. Bottom panel: comparison of VP2 expression in the different lanes. VP2 and β-actin densities were quantified using ImageJ software. Western blot data were semi-quantified and normalized against β-actin protein loading control. Data were obtained from three independent experiments and are presented as means ± SD. **, P < 0.01; ***, P < 0.001.

FIG 9
FIG 9

Disrupting phosphorylation of VP1 reduces viral replication. (A) Schematic model of IBDV rescue. This dual-promoter system for viral rescuing was established in a previous report (18). Briefly, Poly I Pro transcripts minus-sense RNA (segment A and B) are in the forward. In the reverse-ward, CMV promoter transcribes the cap-plus-sense RNA (also acting as mRNA) which directly translates viral proteins. Translated proteins subsequently support the replication of minus-sense RNA to produce plus-sense RNA. Viral proteins and complete double-stranded (dsRNA) could be used to produce infectious IBDV. (B) Cytopathic effect images of DF-1 cells infected (or mock-infected) with WT IBDV and S7A mutant IBDV. Scale bars = 100 μm (frames a, b, and c). DF-1 cells infected (or mock-infected) with WT IBDV and S7A mutant IBDV were detected with anti-VP3 (green) antibody. The nuclei were stained by DAPI. Images were scanned by confocal microscopy with 20-μm scale bars (frames d, e, and f). (C) IBDV titers in the supernatants of 293T cells transfected with the indicated plasmids were determined by TCID50 assay at 72 and 96 h post-transfection. (D) DF-1 cells were infected with WT IBDV and S7A IBDV (1 MOI each) for different durations. Viral genome levels were determined by RT-qPCR. Results were normalized to GAPDH mRNA levels in the same samples. (E) DF-1 cells were infected with WT IBDV and S7A IBDV (1 MOI each) for different durations. Cell lysates were subjected to Western blotting with anti-VP1, anti-VP2, and anti-VP3 antibodies. β-actin was used as the loading control. (F) Comparison of VP1/2/3 expression in the different lanes in panel E. VP1/2/3 and β-actin densities were quantified using ImageJ software. (G) DF-1 cells were infected with 0.1 MOI WT IBDV or S7A IBDV for different durations (12, 18, and 24 h). Viral titers in supernatants of DF-1 cells were determined by TCID50 assay. Western blot data were semi-quantified and normalized against β-actin protein loading control. Data were obtained from three independent experiments and are presented as means ± SD. **, P < 0.01; ***, P < 0.001.

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