Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells
| Autor: | Masaaki Oyama, Yasushi Kawaguchi, Shumpei Tsuda, Hiroko Kozuka-Hata, Akihisa Kato, Zhuoming Liu |
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| Rok vydání: | 2014 |
| Předmět: |
Kinase
viruses Immunology Herpesvirus 1 Human Biology medicine.disease_cause Microbiology Molecular biology Catalysis Serine Multiplicity of infection Herpes simplex virus Viral replication Virology Insect Science medicine Humans Pathogenesis and Immunity Phosphorylation Pyrophosphatases Kinase activity Protein kinase A Protein Kinases |
| Zdroj: | Journal of Virology. 88:655-666 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.02710-13 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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