Bovine herpesvirus-1 US3 Protein Kinase: Critical Residues and Involvement in the Phosphorylation of VP22

J Gen Virol. 2010 May;91(Pt 5):1117-26. doi: 10.1099/vir.0.016600-0. Epub 2009 Dec 16.

Abstract

The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Casein Kinase II / metabolism
  • Cattle
  • Cell Line
  • Chlorocebus aethiops
  • Conserved Sequence
  • Herpesvirus 1, Bovine / enzymology*
  • Immunoprecipitation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Protein Binding
  • Protein Interaction Mapping
  • Protein-Serine-Threonine Kinases / genetics*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Sequence Alignment
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism*
  • Viral Structural Proteins / metabolism*

Substances

  • Viral Proteins
  • Viral Structural Proteins
  • tegument protein VP22, bovine herpesvirus 1
  • Casein Kinase II
  • Protein-Serine-Threonine Kinases
  • US3 protein, Human herpesvirus 1