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Journal of Virology, April 2005, p. 4819-4827, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4819-4827.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of the Varicella-Zoster Virus Gene Product Encoded by Open Reading Frame 35 in Viral Replication In Vitro and in Differentiated Human Skin and T Cells In Vivo

Hideki Ito,^1, Marvin H. Sommer,¹ Leigh Zerboni,¹ Armin Baiker,^1, Bunji Sato,^1, Ruibin Liang,² John Hay,² William Ruyechan,² and Ann M. Arvin^1*

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California,¹ Department of Microbiology & Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, University of Buffalo, Buffalo, New York²

Received 21 September 2004/ Accepted 3 December 2004

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOka35(#1) and rOka35(#2), and replaced ORF35 at a nonnative site, generating two rOka35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOka35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOka35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOka35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOka35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOka35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOka35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.

Present address: Department of Dermatology, Jikei University School of Medicine, 3-19-18 Nishishinbashi Minato, Tokyo, Japan.

Present address: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Munich, Germany.

Present address: Fermentation Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Tokodai, Tsukuba, Ibaraki 300-2698, Japan.

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