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

Sanying Wang; Boli Hu; Weiying Si; Lu Jia; Xiaojuan Zheng; Jiyong Zhou

doi:10.1371/journal.pone.0128828

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

PLoS One. 2015 Jun 5;10(6):e0128828. doi: 10.1371/journal.pone.0128828. eCollection 2015.

Authors

Sanying Wang¹, Boli Hu², Weiying Si³, Lu Jia³, Xiaojuan Zheng⁴, Jiyong Zhou⁵

Affiliations

¹ Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China; Shaoxing Center for Disease Control and Prevention, Shaoxing, PR China.
² College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, PR China.
³ Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China.
⁴ Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China; State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, PR China.
⁵ Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China; College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, PR China; State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, PR China.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Motifs
Animals
Antibodies, Monoclonal / chemistry
Cell Line
Chickens
Cytoskeleton / virology
Epitope Mapping
Fibroblasts / virology*
Gene Expression Regulation, Viral*
HEK293 Cells
Humans
Infectious bursal disease virus / genetics
Infectious bursal disease virus / metabolism
Isoelectric Point
Molecular Sequence Data
Mutagenesis, Site-Directed
Phosphoproteins / genetics*
Phosphoproteins / metabolism
Phosphorylation
Protein Precursors / genetics*
Protein Precursors / metabolism
Proteolysis
Viral Structural Proteins / genetics*
Viral Structural Proteins / metabolism

Substances

Antibodies, Monoclonal
Phosphoproteins
Protein Precursors
VP4 protein, infectious bursal disease virus
Viral Structural Proteins

Grants and funding

This work was supported by grants from China Agriculture Research System (Grant No. CARS-41-K11) and National Natural Science Foundation of China (Grant No. 30870117 and 30800825). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.