title: Herpes simplex virus 1 protein kinase Us3 phosphorylates viral dUTPase and regulates its catalytic activity in infected cells.
abstract: Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase.
authors.affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Akihisa
authors.initials: A
authors.lastname: Kato
authors.forename: Shumpei
authors.initials: S
authors.lastname: Tsuda
authors.forename: Zhuoming
authors.initials: Z
authors.lastname: Liu
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kozuka-Hata
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oyama
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawaguchi
mesh_terms: D002384:Catalysis; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism*
chemical_list: D011494:Protein Kinases; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase