Herpes simplex virus (HSV) entry into cells requires the binding of HSV glycoprotein D (gD) to one of several cell surface receptors. The crystal structure of gD complexed with one of these receptors, HveA/HVEM, was recently determined (21). HveA is a member of the TNF receptor family and contains four characteristic cysteine rich domains (CRDs). The core of gD is comprised of an immunoglobulin fold flanked by a long C-terminal extension and an N-terminal hairpin loop. Fourteen amino acids within the gD N-terminal loop directly contact seventeen amino acids in HveA CRD1 and four amino acids in CRD2.
In this dissertation, I present a structure-based dissection of the gD-HveA complex. To determine the contribution of individual residues to virus entry, each gD and HveA contact residue was mutated independently. I assessed the ability of each mutant protein to form a complex with its wild-type partner and to mediate HSV entry and cell-cell fusion. Mutations in most of the contact residues of gD and HveA had little or no effect on binding and entry. Notably, for each protein, a few residues played a key role in the interaction.
Three critical regions at the gD-HveA interface were identified. (i) HveA-Y23 protrudes into a crevice on the surface of gD at the center of the interface and represents a "hot spot" for gD binding. (ii) An intermolecular β-sheet formed between gD residues 27-29 and HveA residues 35-37 is critical for virus entry. (iii) Although the functional gD binding site lies primarily within HveA CRD1, two residues within CRD2 contribute to complex formation. These three regions may represent good targets for the design of inhibitors to block the gD-receptor interaction.
In addition, preliminary studies examined the mechanism by which gD undergoes a conformational change upon HveA binding. Unexpectedly, these studies led to the identification of a potential binding site on gD for nectin-1, another HSV receptor. Finally, a comparison of the receptor binding properties of three alphaherpesvirus gD homologues revealed that virus entry occurs over a wide range of affinities.
