title: Structure-based mutagenesis of herpes simplex virus glycoprotein D defines three critical regions at the gD-HveA/HVEM binding interface.
abstract: Herpes simplex virus (HSV) entry into cells requires the binding of glycoprotein D (gD) to one of several cell surface receptors. The crystal structure of gD bound to one of these receptors, HveA/HVEM, reveals that the core of gD comprises an immunoglobulin fold flanked by a long C-terminal extension and an N-terminal hairpin loop. HveA is a member of the tumor necrosis factor receptor family and contains four cysteine-rich domains (CRDs) characteristic of this family. Fourteen amino acids within the gD N-terminal loop comprise the entire binding site for HveA. To determine the contribution of each gD contact residue to virus entry, we constructed gD molecules mutated in these amino acids. We determined the abilities of the gD mutants to bind receptors, facilitate virus entry, and mediate cell-cell fusion. Seven of the gD mutants exhibited wild-type levels of receptor binding and gD function. Results from the other seven gD mutants revealed three critical regions at the gD-HveA interface. (i) Several gD residues that participate in an intermolecular beta-sheet with HveA were found to be crucial for HveA binding and entry into HveA-expressing cells. (ii) Two gD residues that contact HveA-Y23 contributed to HveA binding but were not required for mediating entry into cells. HveA-Y23 fits into a crevice on the surface of gD and was previously shown to be essential for gD binding. (iii) CRD2 was previously shown to contribute to gD binding, and this study shows that one gD residue that contacts CRD2 contributes to HveA binding. None of the gD mutations prevented interaction with nectin-1, another gD receptor. However, when cotransfected with the other glycoproteins required for fusion, two gD mutants gained the ability to mediate fusion of cells expressing nectin-2, a gD receptor that interacts with several laboratory-derived gD mutants but not with wild-type gD. Thus, results from this panel of gD mutants as well as those of previous studies (A. Carfi, S. H. Willis, J. C. Whitbeck, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and D. C. Wiley, Mol. Cell 8:169-179, 2001, and S. A. Connolly, D. J. Landsburg, A. Carfi, D. C. Wiley, R. J. Eisenberg, and G. H. Cohen, J. Virol. 76:10894-10904, 2002) provide a detailed picture of the gD-HveA interface and the contacts required for functional interaction. The results demonstrate that of the 35 gD and HveA contact residues that comprise the gD-HveA interface, only a handful are critical for complex formation.
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. sconnoll@med.upenn.edu
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Connolly
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Landsburg
authors.forename: Andrea
authors.initials: A
authors.lastname: Carfi
authors.forename: Don C
authors.initials: DC
authors.lastname: Wiley
authors.forename: Gary H
authors.initials: GH
authors.lastname: Cohen
authors.forename: Roselyn J
authors.initials: RJ
authors.lastname: Eisenberg
mesh_terms: D000818:Animals; D001665:Binding Sites; D002459:Cell Fusion; D002460:Cell Line; D003460:Crystallization; D004797:Enzyme-Linked Immunosorbent Assay; D015726:Giant Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000378:metabolism*; D053240:Receptors, Tumor Necrosis Factor, Member 14; D011991:Receptors, Virus / Q000737:chemistry / Q000378:metabolism*; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D018124:Receptors, Tumor Necrosis Factor; D053240:Receptors, Tumor Necrosis Factor, Member 14; D011991:Receptors, Virus; C506028:TNFRSF14 protein, human; C506029:Tnfrsf14 protein, mouse; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1