Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22.
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.
Labiuk
Shaunivan L
SL
Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada.
Lobanov
Vladislav
V
Lawman
Zoe
Z
Snider
Marlene
M
Babiuk
Lorne A
LA
van Drunen Littel-van den Hurk
Sylvia
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D047390:Casein Kinase II / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D007242:Herpesvirus 1, Bovine / Q000201:enzymology*; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*
D014764:Viral Proteins; D015678:Viral Structural Proteins; C434023:tegument protein VP22, bovine herpesvirus 1; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; C095207:US3 protein, Human herpesvirus 1