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Journal of Virology, January 2008, p. 700-709, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.02192-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Engineered Disulfide Bonds in Herpes Simplex Virus Type 1 gD Separate Receptor Binding from Fusion Initiation and Viral Entry

Eric Lazear,^1* Andrea Carfi,⁴ J. Charles Whitbeck,¹ Tina M. Cairns,¹ Claude Krummenacher,² Gary H. Cohen,¹ and Roselyn J. Eisenberg³

Department of Microbiology,¹ Biochemistry, School of Dental Medicine,² Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,³ Department of Biochemistry, IRBM P Angeletti, I-00040 Pomezia (Rome), Italy⁴

Received 5 October 2007/ Accepted 1 November 2007

Glycoprotein D (gD) is the receptor binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, herpesvirus entry mediator (HVEM) and nectin-1. While both receptor binding regions are found within the first 234 amino acids, a crystal structure shows that the C terminus of the gD ectodomain normally occludes the receptor binding sites. Receptor binding must therefore displace the C terminus, and this conformational change is postulated to be required for inducing fusion via gB and gH/gL. When cysteine residues are introduced at positions 37 and 302 of gD, a disulfide bond is formed that stabilizes the C terminus and prevents binding to either receptor. We speculated that if disulfide bonds were engineered further upstream, receptor binding might be separated from the induction of fusion. To test this, we made five additional double cysteine mutants, each potentially introducing a disulfide bond between the ectodomain C terminus and the core of the gD ectodomain. The two mutants predicted to impose the greatest constraint were unable to bind receptors or mediate cell-cell fusion. However, the three mutants with the most flexible C terminus bound well to both HVEM and nectin-1. Two of these mutants were impaired in cell-cell fusion and null-virus complementation. Importantly, a third mutant in this group was nonfunctional in both assays. This mutant clearly separates the role of gD in triggering fusion from its role in receptor binding. Based upon the properties of the panel of mutants we conclude that fusion requires greater flexibility of the gD ectodomain C terminus than does receptor binding.

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* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania, 240 S. 40th St., Levy Rm. 233, Philadelphia, PA 19104. Phone: (215) 898-6553. Fax: (215) 898-8385. E-mail: lazear{at}mail.med.upenn.edu

Published ahead of print on 21 November 2007.

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Journal of Virology, January 2008, p. 700-709, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.02192-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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• Gianni, T., Amasio, M., Campadelli-Fiume, G. (2009). Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain. J. Biol. Chem. 284: 17370-17382 [Abstract] [Full Text]