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Journal of Virology, January 2005, p. 1232-1243, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.1232-1243.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Phosphorylation Site Mutations Affect Herpes Simplex Virus Type 1 ICP0 Function

David J. Davido,1 William F. von Zagorski,1 William S. Lane,2 and Priscilla A. Schaffer1*

Departments of Medicine and Microbiology and Molecular Genetics, Harvard Medical School at Beth Israel Deaconess Medical Center, Boston,1 Microchemistry and Proteomics Analysis Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts2

Received 16 June 2004/ Accepted 28 July 2004

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.

* Corresponding author. Mailing address: Beth Israel Deaconess Medical Center, 330 Brookline Ave., RN 123, Boston, MA 02215. Phone: (617) 667-2958. Fax: (617) 667-8540. E-mail: pschaffe{at}bidmc.harvard.edu.

Journal of Virology, January 2005, p. 1232-1243, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.1232-1243.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Boutell, C., Everett, R., Hilliard, J., Schaffer, P., Orr, A., Davido, D. (2008). Herpes Simplex Virus Type 1 ICP0 Phosphorylation Mutants Impair the E3 Ubiquitin Ligase Activity of ICP0 in a Cell Type-Dependent Manner. J. Virol. 82: 10647-10656 [Abstract] [Full Text]  
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