Structure-based analysis of the herpes simplex virus glycoprotein D binding site present on herpesvirus entry mediator HveA (HVEM)

Abstract

Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular beta-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.

FIG. 1.

Diagram of full-length HveA and gD. The amino acid numbers begin with the first amino acid in the mature protein after signal sequence cleavage. The positions of N-glycosylation sites (lollipops) and transmembrane regions (TM) are indicated. The HveA amino acids comprising each of the four CRDs are labeled. The gD amino acids comprising each of four defined functional regions (FR) and the group VII MAb epitope (gray circle) are labeled. The disulfide bond pattern (dotted lines) and locations of cysteines (C) within gD are indicated. Arrows indicate the sites of truncation for the proteins used to solve the crystal structure.

FIG. 2.

Highlighted regions within the gD-HveA crystal structure. (A) Ribbon diagram of the crystal structure of HveA bound to gD. The N- and C-terminal residues observed in the crystal structure and the location of HveA CRDs are indicated. The HveA molecule is shown partially in blue, and the HveA contact residues found within CRD1 and CRD2 are shown in green. The gD molecule is shown in gray, with the contact residues located in the N-terminal loop of gD shown in red. Contact residues were defined as amino acids containing atoms that come within 4 Å of the partner molecule (see Table 1). Some of the contact residues are numbered for reference. (B) An enlarged view of the gD-HveA interface shown in the same orientation as in panel A. HveA contact residues are displayed in green, and gD contact residues are red. The boxed area indicates a region shown at higher magnification in panel D. (C) Magnification of a disulfide bond within HveA CRD1 and three β-strands within the gD-HveA binding site. HveA-C37 (yellow) forms a disulfide bond with HveA-C19 (purple). Residues 35 to 37 within a β-strand of HveA (residues 35 to 40, shown in green) form hydrogen bonds with a short β-strand on gD (gD residues 27 to 29, shown in red) to form an intermolecular, antiparallel β-sheet. This augments a two-stranded β-sheet in HveA CRD1 formed by residues 22 to 26 (blue) and residues 35 to 40 (green). (D) Interactions between HveA-Y23 and gD amino acids. Carbon (green), oxygen (red), nitrogen (purple), and sulfur (yellow) atoms are shown. The blue dotted lines indicate hydrogen bonds between HveA-Y23 and gD. The black lines indicate other interactions, defined as distances of 4 Å or less (see Table 1). The hydroxyl group of HveA-Y23 interacts with gD-A12 and gD-L25. The phenyl ring of HveA-Y23 interacts with gD-M11 and gD-A12. (E) Space-filling model of the gD binding site on the surface of HveA. The gD-HveA interface from panel B is rotated so that the N-terminal loop of gD lies on top of the HveA binding surface. The N-terminal loop of gD is shown as a gray ribbon, with the gD contact residues colored red. The HveA contact residues are numbered. The space-filling spheres represent atoms of HveA contact residues from category 1 (blue), category 2 (purple), category 3 (yellow), and category 4 (green) (see Table 1). HveA-Y23 (green) is located at the center of the interface.

FIG. 3.

Expression of HveA mutant proteins on transfected cells. B78-H1 cells were transiently transfected with plasmids carrying each of the HveA mutants and evaluated for the expression of HveA after 48 h. (A) Cells were stained with fluorescently labeled anti-HveA PAb IgG and visualized by immunofluorescence microscopy to detect cell surface expression. Results for the transfection of wild-type HveA (wtHveA) and three representative HveA mutants are shown. (B) HveA expression was quantitated by CELISA. Cells were plated on 96-well plates and titrated with anti-HveA PAb IgG (R140) to estimate the level of cell surface expression. Sample data are shown for wild-type HveA and three representative HveA mutants. (C) Cells were stained with fluorescently labeled (Alexa Fluor 488) anti-HveA PAb IgG and analyzed by flow cytometry (FACS). Sample data for wild-type HveA and three representative HveA mutants show the level and range of positive staining. (D) Flow cytometry data for all of the HveA mutants are shown to indicate the percentage of cells staining positive for HveA. The dotted line represents 50% of the cells staining positive for HveA. Mutants were divided into four categories based on their ability to bind gD (see Fig. 4 and Table 1).

FIG. 4.

Binding of a truncated form of gD, gD(285t), to HveA proteins expressed on transfected cells. Transiently transfected B78-H1 cells expressing wild-type HveA and the HveA mutants were seeded on 96-well plates, incubated with different concentrations of gDt, washed, and probed with an anti-gD PAb to detect the level of gDt binding. (A) Sample data are shown for wild-type HveA (wtHveA) and three representative HveA mutants. Assays detecting HveA expression were run in parallel (Fig. 3). (B) Data for all of the HveA mutant proteins binding gDt at one concentration, 100 nM gD(285t), are shown to illustrate the basis for the separation of the HveA mutants into reduced (striped bars), wild-type (black bars), and increased (crosshatched bars) binding categories. gDt binding is expressed as a percentage of gDt binding to wild-type HveA. Results from one representative experiment are shown. (C) Data for HveA mutants binding gDt at a higher concentration, 1 μM gD(285t), are shown to illustrate the basis for the separation of the HveA mutants into negative (white bars) and reduced (striped bars) categories.

FIG. 5.

HveA mutant proteins mediating HSV entry into transfected cells. Transiently transfected B78-H1 cells expressing the HveA mutant proteins were seeded on 96-well plates, incubated with an HSV β-galactosidase reporter virus for 6 h, and assayed for β-galactosidase activity as a measure of virus entry. (A) Sample data show the ability of wild-type HveA (wtHveA) and three representative HveA mutants to mediate entry of increasing amounts of HSV. Assays detecting HveA expression and gDt binding were run in parallel (Fig. 3 and 4). (B) Data show the original panel of 21 HveA mutants mediating entry at one concentration of HSV. Mutants were divided into the categories based on gD binding phenotype (see Fig. 4 and Table 1). Data are expressed as a percentage of entry mediated by wild-type HveA. Results from one representative experiment are shown. (C) The ability of additional HveA mutants to mediate entry of increasing amounts of HSV is shown. (D) Sample data show the inability of wild-type HveA and three representative HveA mutants to mediate entry of increasing amounts of a mutant β-galactosidase reporter virus, HSV Rid1, after 6 h. Vero cells were included as a positive control for HSV Rid1 entry.

FIG. 6.

Ability of HveA mutant proteins to oligomerize. Extracts of 293T cells transfected with HveA mutants were run on SDS-PAGE under nondenaturing conditions, transferred to nitrocellulose, and probed with an anti-HveA PAb. Results for wild-type HveA (wtHveA) and four selected HveA mutant proteins are shown. The positions of molecular size markers are shown (in kilodaltons), along with the expected positions of the HveA monomer and dimer.