title: Regulation and function of phosphorylation on VP8, the major tegument protein of bovine herpesvirus 1.
abstract: The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study, VP8 was found to be phosphorylated in both transfected and infected cells but was detected as a nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by US3 and CK2 were further analyzed. An in vitro kinase assay, site-directed mutagenesis, and liquid chromatography-mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S(16) is a primary phosphoreceptor for US3, and it subsequently triggers phosphorylation at S(32). CK2 has multiple active sites, among which T(107) appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated VP8 mutant was constructed and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing PML protein and that this function depends on the phosphorylation of VP8.
  
  
  OBJECTIVE
  The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S(16) initiates further phosphorylation at S(32) by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T(107) having the highest level of phosphorylation. Evidence for a difference in the phosphorylation status of VP8 in host cells and mature virus is presented for the first time. Phosphorylation was found to be a critical modification, which enables VP8 to attract and to redistribute PML protein in the nucleus. This might promote viral replication through interference with a PML-mediated antiviral defense. This study provides new insights into the regulation of VP8 phosphorylation and suggests a novel, phosphorylation-dependent function for VP8 in the life cycle of BoHV-1, which is important in view of the fact that VP8 is essential for virus replication in vivo.
authors: VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors: Kuan
authors: K
authors: Zhang
authors: VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors: Sharmin
authors: S
authors: Afroz
authors: VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors: Robert
authors: R
authors: Brownlie
authors: VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors: Marlene
authors: M
authors: Snider
authors: VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada sylvia.vandenhurk@usask.ca.
authors: Sylvia
authors: S
authors: van Drunen Littel-van den Hurk
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D047390:Casein Kinase II / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle / Q000821:virology*; D002853:Chromatography, Liquid; D007242:Herpesvirus 1, Bovine / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014771:Virion / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D025521:Tumor Suppressor Proteins; C442561:VP8 protein, Bovine herpesvirus 1; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases