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Journal of Virology, August 2008, p. 7653-7665, Vol. 82, No. 15
0022-538X/08/$08.00+0     doi:10.1128/JVI.00311-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Varicella-Zoster Virus Open Reading Frame 66 Protein Kinase Is Required for Efficient Viral Growth in Primary Human Corneal Stromal Fibroblast Cells

Angela Erazo,^1,2, Michael B. Yee,^2, Nikolaus Osterrieder,⁴ and Paul R. Kinchington^2,3*

Graduate Program in Molecular Virology and Microbiology,¹ Departments of Ophthalmology,² Molecular Microbiology and Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania,³ Department of Microbiology and Immunology, Cornell University, Ithaca, New York⁴

Received 12 February 2008/ Accepted 13 May 2008

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.

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* Corresponding author. Mailing address: Department of Ophthalmology, 1020 EEI building, 203 Lothrop Street, University of Pittsburgh, Pittsburgh, PA 15213. Phone: (412) 647-6319. Fax: (412) 647-5880. E-mail: kinchingtonp{at}upmc.edu

Published ahead of print on 21 May 2008.

A.E. and M.B.Y. contributed equally to this work.

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Journal of Virology, August 2008, p. 7653-7665, Vol. 82, No. 15
0022-538X/08/$08.00+0     doi:10.1128/JVI.00311-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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