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Journal of Virology, February 2006, p. 1710-1723, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1710-1723.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Phosphorylation of the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein IE62 by the VZV Open Reading Frame 66 Protein Kinase

Amie J. Eisfeld,3,{dagger} Stephanie E. Turse,1,{dagger} Sara A. Jackson,4 Edwina C. Lerner,2 and Paul R. Kinchington1,2*

Departments of Ophthalmology,1 Molecular Genetics and Biochemistry,2 Graduate Programs in Molecular Virology and Microbiology,3 Biochemistry and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 152134

Received 21 July 2005/ Accepted 22 November 2005

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

* Corresponding author. Mailing address: 1020 Eye & Ear Institute, University of Pittsburgh, 203 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 647-6319. Fax: (412) 647-5880. E-mail: Kinchingtonp{at}upmc.edu.

{dagger} The first two authors contributed equally to this work.

Journal of Virology, February 2006, p. 1710-1723, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1710-1723.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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