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Journal of Virology, May 2005, p. 5889-5899, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.5889-5899.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

High-Level Expression of Marek's Disease Virus Glycoprotein C Is Detrimental to Virus Growth In Vitro

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853,¹ Laboratoire de Virologie Moléculaire et Immunologie, UR 086 BASE, Centre de Recherches INRA de Tours, 37380 Nouzilly, France,² Lohmann Animal Health, 27472 Cuxhaven, Germany³

Received 26 October 2004/ Accepted 28 December 2004

Expression levels of Marek's disease virus (MDV) glycoprotein C (gC) are significantly reduced after serial virus passage in cell culture. Reduced gC expression coincides with enhanced MDV growth in vitro and attenuation. To analyze this phenomenon in detail, a full-length infectious MDV clone was modified by Red-based and shuttle mutagenesis in Escherichia coli. Besides a gC-negative deletion mutant harboring a kanamycin resistance gene, a markerless mutant with the U_L44 gene deleted was constructed. On the basis of this deletion mutant, the original or a modified U_L44 gene with a mutated start codon (AUGACG) was reinserted into the authentic locus. Similarly, mutants expressing authentic gC or the start codon mutation under the control of a strong constitutive promoter were generated. In vitro studies demonstrated that gC deletion mutants induced twofold-larger plaques than the parental virus did, whereas constitutive overexpression of the glycoprotein resulted in a more than twofold reduction in plaque size. In addition, plaque sizes of the gC deletion mutant were reduced when virus was grown using supernatants from cells infected with parental virus, but supernatants obtained from cells infected with the gC deletion mutant had no measurable effect on plaque size. The results indicated that (i) expression of MDV gC, albeit at low levels in a highly passaged virus, had a significant negative impact on the cell-to-cell spread capabilities of the virus, which was alleviated in its absence and exacerbated by its overexpression, and that (ii) this activity was mediated by the secreted form of MDV gC.

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