Avibirnavirus VP4 Protein Is a Phosphoprotein and Partially Contributes to the Cleavage of Intermediate Precursor VP4-VP3 Polyprotein

<div><p>Birnavirus-encoded viral protein 4 (VP4) utilizes a Ser/Lys catalytic dyad mechanism to process polyprotein. Here three phosphorylated amino acid residues Ser538, Tyr611 and Thr674 within the VP4 protein of the infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, were identified by mass spectrometry. Anti-VP4 monoclonal antibodies finely mapping to phosphorylated (p)Ser538 and the epitope motif ⁵³⁰PVVDGIL⁵³⁶ were generated and verified. Proteomic analysis showed that in IBDV-infected cells the VP4 was distributed mainly in the cytoskeletal fraction and existed with different isoelectric points and several phosphorylation modifications. Phosphorylation of VP4 did not influence the aggregation of VP4 molecules. The proteolytic activity analysis verified that the pTyr611 and pThr674 sites within VP4 are involved in the cleavage of viral intermediate precursor VP4-VP3. This study demonstrates that IBDV-encoded VP4 protein is a unique phosphoprotein and that phosphorylation of Tyr611 and Thr674 of VP4 affects its serine-protease activity.</p></div

LC-MS/MS identification of phosphorylation sites within VP4 of IBDV.

<p>Note: The bold rows revealed the identified peptides that contained a phosphorylated amino acid.</p><p>^“#” is the phosphorylated amino acid residues.</p><p>^“@” indicates methylation of Methionine (M).</p><p>All proteins listed in the table were found to have a statistically significant p-value of less than 0.05.</p><p>LC-MS/MS identification of phosphorylation sites within VP4 of IBDV.</p

Primers for chicken CD132-dependent cytokines and β-actin in the method of Real-time PCR.

<p>Primers for chicken CD132-dependent cytokines and β-actin in the method of Real-time PCR.</p

Identification of anti-chCD132 mAb bound to cellular CD132 on the SMC surface.

<p>(A) SDS-PAGE analysis of <i>Escherichia coli</i>-expressed chCD132; M, molecular weight marker; lane 1, bacterial lysates of <i>E. coli</i> BL21 (DE3) transformed with pET28a; lane 2, bacterial lysates containing rchCD132. (B) Western blot analysis of rchCD132 recognized by 6 anti-chCD132 mAbs. (C) anti-chCD132 mAb C10 recognized by chCD132 expressed on the SMC surface using indirect immunofluorescencestaining (×10).</p

VP4 is a complex with different isoelectric points in IBDV-infected cells.

<p>(A) Western blot analysis of subcellular fractionated IBDV-infected DF-1 cells. The cells were infected with IBDV at the MOI of 1, harvested at 24 h post-infection and centrifuged at 8,000 × <i>g</i> for 5 min. The cytosolic, membrane, nuclear and cytoskeletal fractions of IBDV-infected (+) or mock-infected (-) DF-1 cells were sequentially isolated using the Qproteome Cell Compartment Kit. Equivalent amounts of each fraction (20 μg) were subjected to 12% SDS-PAGE followed by Western blot analysis. GAPDH, calnexin, histone H3 and β-actin were used as markers of the cytosolic, cell membrane, nuclear and cytoskeletal fractions, respectively. VP4 was detected with the anti-pSer538 or P530 mAb. Averaged densitometric intensities of three replicate immunoblots are shown. P530: VP4 protein recognized with the P530 mAb; pSer538: VP4 protein reacted with the pSer538 mAb. Error bar represents the standard deviation. (B) 2-DE and 2-DE blot analysis. Upper panel: Protein (200 μg) from the cytoskeletal fraction of IBDV-infected DF-1 cells were separated by 2-DE and visualized by colloidal Coomassie blue staining. Middle panel: Five VP4 protein spots recognized by the P530 mAb in a 2-DE blot. Lower panel: Two phosphorylated VP4 protein spots reacted with the pSer538 mAb in a 2-DE blot. (C) VP4 protein in IBDV-infected DF-1 cells was immunoprecipitated and detected with the P530 and pSer538 mAbs, respectively. (D) Co-localization of phosphorylated and unphosphorylated VP4 protein. Direct immunofluorescence assay of VP4 protein in IBDV-infected DF-1 cells with pSer538 and P530 mAbs. DF-1 cells were infected with IBDV, and fixed cells were probed with Alexa Fluor 555-labeled pSer538 (red) and Alexa Fluor 488-labeled P530 (green) mAbs. Nuclei were counterstained with DAPI. Overlapping signals were revealed by detection of VP4 by the P530 and pSer538 mAbs (merge).</p

The summary of the primers used in this study.

<p>Note: Italic and bold indicate mutation base.</p><p>The summary of the primers used in this study.</p

The summary of proteolytic cleavage result affected by a series of substitution in this study.

<p>Note:</p><p>^“+” means such mutation does not affect intermediate precursor VP4-VP3 cleavage;</p><p>^“-” means such mutation affects intermediate precursor VP4-VP3 cleavage.</p><p>The summary of proteolytic cleavage result affected by a series of substitution in this study.</p

Synthetic peptides of the IBDV VP4 protein in this study.

<p>Note:</p><p>“*” indicates the amino acids with phosphorylation.</p><p>The letter “c” means cysteine appended to the Sulfo-SMCC cross-linker. Mutated residues are underlined.</p><p>Synthetic peptides of the IBDV VP4 protein in this study.</p

Analysis of proteolytic activity of viral VP4 protein.

<p>(A) 293T cells were transfected with the recombinant wild-type A-segment plasmid or the Ala or Asp substituted A-segment plasmids at sites pSer538, pTyr611 and pThr674 within VP4. At 24 h post-transfection, cell samples were harvested and electrophoresed on 12% SDS-PAGE gels for Western blot analysis with mAbs specific to VP3 and VP4 proteins. GAPDH was used as a loading control. (B) Recombinant plasmids used in (A) were translated with the TNT T7 Quick Coupled Transcription/Translation System, and expressed proteins were detected with mAbs specific for VP3 and VP4 proteins. (C) Analysis of co-localization between IBDV-encoding proteins within segment A. 293T cells were transfected with the IBDV A-segment mutant with the single dephospho- and phospho-mimicking VP4 gene. Wild-type IBDV A-segment transfected cells were used as a positive control. At 24 h post-transfection, the cells were fixed and probed with chicken anti-VP2 pAb, mouse anti-VP3 mAb and rabbit anti-VP4 pAb followed by FITC-conjugated goat anti-chicken IgG (green), Alexa Fluor 647 donkey anti-mouse IgG (blue) and Alexa Fluor 546 donkey ant-rabbit IgG (red). Nuclei were counterstained with DAPI (grey). The cells were observed with a laser Zeiss LSM510 laser confocal microscope. Cells transfected with the A segment with the Tyr611Asp and Thr674 Ala/Asp substitutions revealed co-localization between the IBDV-encoded proteins.</p

Fine mapping of epitopes on IBDV VP4 protein with peptide ELISA and peptide dot-ELISA.

<p>(A) Eleven BSA-conjugated peptides (spanning residues 515–558, 550–565, 563–600, 598–631, 618–641, 638–662, 653–674, 680–706, 700–717, 713–741 and 720–750) were coated on a 96-well plate in the peptide ELISA or dotted on a nitrocellulose membrane in the peptide dot-ELISA and probed with mAbs 4A8, 5B2, 5C7, 7A4 and 7H8 to the IBDV VP4 protein. The mAbs 7A4 and 7H8 could react with Pep515-558 but not the other peptides, and the mAbs 4A8, 5B2 and 5C7 did not react with any of these peptides. (B) The epitope motif of 7A4 and 7H8 was localized within the residues 530–536 by the peptide ELISA, and residues of ⁵³⁰Pro and ⁵³³DGIL⁵³⁶ were indispensable for forming the antigenic epitope. (C) BSA-conjugated phosphorylated and unphosphorylated peptides spanning residues 533-549pSer538, 533–549, 602-619pTyr611, 602–619, 667-683pThr674, 667–683 were coated on a 96-well plate in the peptide ELISA or dotted on a nitrocellulose membrane in a peptide dot-ELISA and probed with mAbs 4A8, 5B2 and 5C7 to the IBDV VP4 protein. The mAbs 4A8, 5B2 and 5C7 could recognize specifically the same phosphorylated peptide 533-549pSer538 but not unphosphorylated peptide 533–549.</p