Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22

Abstract

The U S 3 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.

Cell Cycle Modulation by Marek’s Disease Virus: The Tegument Protein VP22 Triggers S-Phase Arrest and DNA Damage in Proliferating Cells

“…Overexpression of UL13 and/or US3 in low proliferating cells (CESC) or high proliferating cells (LMH) had no effect on the cell cycle, thus excluding a direct involvement of these kinases in the cell cycle modulation. However, pUS3 and pUL13 are able to phosphorylate various cellular and viral proteins, including the VP22 proteins encoded by HSV-1 and -2, as well as BoHV-1 [63], [64], [65]. So far, the phosphorylation status of MDV-VP22 during infection has not been investigated, and we cannot exclude post-translational modifications of MDV-VP22 by UL13 and/or US3, as previously shown for other alphaherpesviruses.…”

Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation

“…Antibodies. Monoclonal antibodies specific for gB, gC, and gD (21) and polyclonal antibodies specific for VP8 (9), VP22 (22), VP5 (8), and US3 (22) were raised as described previously. A monoclonal anti-Golgi apparatus 58,000-molecular-weight (58K) protein antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).…”

US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells

“…However, the kinase activity of US3 is unlikely to be affected because the catalytic loop and the ATP-binding pocket are conserved ( Fig. 3) (17). These results imply that US3 is essential for cytoplasmic localization of VP8 at a later stage during BoHV-1 infection.…”

“…US3 was required for cytoplasmic localization of VP8. To determine the reason for this observation, the translocation of wild-type VP8 was examined in transfected cells expressing either wild-type US3 or a mutated US3 (US3-K282E) (17), which does not phosphorylate VP8 (8). VP8 was mainly localized in the nucleus when US3 was absent or mutated ( Fig.…”