@article{mbs:/content/journal/jgv/10.1099/vir.0.016600-0, author = "Labiuk, Shaunivan L. and Lobanov, Vladislav and Lawman, Zoe and Snider, Marlene and Babiuk, Lorne A. and van Drunen Littel-van den Hurk, Sylvia", title = "Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22", journal= "Journal of General Virology", year = "2010", volume = "91", number = "5", pages = "1117-1126", doi = "https://doi.org/10.1099/vir.0.016600-0", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.016600-0", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", 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.", }