title: Engineered disulfide bonds in herpes simplex virus type 1 gD separate receptor binding from fusion initiation and viral entry.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Pennsylvania, 240 S. 40th St., Levy Rm. 233, Philadelphia, PA 19104, USA. lazear@mail.med.upenn.edu
authors.forename: Eric
authors.initials: E
authors.lastname: Lazear
authors.forename: Andrea
authors.initials: A
authors.lastname: Carfi
authors.forename: J Charles
authors.initials: JC
authors.lastname: Whitbeck
authors.forename: Tina M
authors.initials: TM
authors.lastname: Cairns
authors.forename: Claude
authors.initials: C
authors.lastname: Krummenacher
authors.forename: Gary H
authors.initials: GH
authors.lastname: Cohen
authors.forename: Roselyn J
authors.initials: RJ
authors.lastname: Eisenberg
mesh_terms: D019943:Amino Acid Substitution; D004220:Disulfides / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology*; D053585:Virus Attachment; D053586:Virus Internalization
chemical_list: D004220:Disulfides; D014759:Viral Envelope Proteins; C515577:glycoprotein gD, herpes simplex virus type 1