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

Sanying Wang,Weiying Si,Boli Hu,Lu Jia,Jiyong Zhou,Xiaojuan Zheng,José R Caston

doi:10.1371/journal.pone.0128828

Abstract

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.

Highlights

Infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, damages the precursors of antibody-producing B lymphocytes in the bursa of Fabricius and causes severe immunosuppression and mortality in young chickens
The viral protein 4 (VP4) protein generated in IBDV-infected DF-1 cells was confirmed by Western blot analysis using the anti-VP4 pAb (S1 Fig), and the specific protein band was gel-purified after separation by 12% SDS-PAGE
Western blot analysis showed that these generated monoclonal antibodies (mAbs) could react with VP4 protein expressed in both VP4-transfected and IBDV-infected DF-1 cells (Fig 1A)

Summary

Introduction

Infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, damages the precursors of antibody-producing B lymphocytes in the bursa of Fabricius and causes severe immunosuppression and mortality in young chickens. The IBDV genome is characterized by a bisegmented double-stranded RNA (segments A and B). The smaller segment B only encodes the VP1 with a molecular weight of 90 kDa. VP1 is the putative RNA-dependent RNA polymerase which interacts with the viral genome [1, 2] and is involved in IBDV mRNA translation via association with the carboxy-terminal domain of the eukaryotic translation initiation factor 4AII [3]. It has been demonstrated to affect viral replication kinetics and modulate the virulence [4,5,6]. The larger segment A contains two partially

Methods

Results

Conclusion