JVI Accepts, published online ahead of print on 21 November 2007

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J. Virol. doi:10.1128/JVI.02192-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Engineered disulfide bonds in herpes simplex virus type 1 gD separate receptor binding from fusion initiation and viral entry

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

Department of Microbiology and of Biochemistry, School of Dental Medicine and Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. Department of Biochemistry, IRBM P Angeletti, I-00040 Pomezia (Rome), Italy

^* To whom correspondence should be addressed. Email: lazear{at}mail.med.upenn.edu.

Glycoprotein D (gD) is the receptor-binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, 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 non-functional 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 fusion requires greater flexibility of the gD ectodomain C-terminus than does receptor binding.

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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]