Amino acid substitutions in the V domain of nectin-1 (HveC) that impair entry activity for herpes simplex virus types 1 and 2 but not for Pseudorabies virus or bovine herpesvirus 1

Abstract

The entry of herpes simplex virus (HSV) into cells requires the interaction of viral glycoprotein D (gD) with a cellular gD receptor to trigger the fusion of viral and cellular membranes. Nectin-1, a member of the immunoglobulin superfamily, can serve as a gD receptor for HSV types 1 and 2 (HSV-1 and HSV-2, respectively) as well as for the animal herpesviruses porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1). The HSV-1 gD binding domain of nectin-1 is hypothesized to overlap amino acids 64 to 104 of the N-terminal variable domain-like immunoglobulin domain. Moreover, the HSV-1 and PRV gDs compete for binding to nectin-1. Here we report that two amino acids within this region, at positions 77 and 85, are critical for HSV-1 and HSV-2 entry but not for the entry of PRV or BHV-1. Replacement of either amino acid 77 or amino acid 85 reduced HSV-1 and HSV-2 gD binding but had a lesser effect on HSV entry activity, suggesting that weak interactions between gD and nectin-1 are sufficient to trigger the mechanism of HSV entry. Substitution of both amino acid 77 and amino acid 85 in nectin-1 significantly impaired entry activity for HSV-1 and HSV-2 and eliminated binding to soluble forms of HSV-1 and HSV-2 gDs but did not impair the entry of PRV and BHV-1. Thus, amino acids 77 and 85 of nectin-1 form part of the interface with HSV gD or influence the conformation of that interface. Moreover, the binding sites for HSV and PRV or BHV-1 gDs on nectin-1 may overlap but are not identical.

FIG. 1.

Alignment of nectin-1 and nectin-2 amino acid sequences in the N-terminal V domain. The amino acid sequence contained between the two conserved cysteines of the V domain of nectin-1 is aligned below the corresponding sequence of nectin-2. Numbers shown above the amino acids correspond to their positions from the initial methionine. Regions in nectin-2 previously shown to be critical for HSV entry activity and referred to as regions A and B are indicated by single underlining (23). The asterisks indicate the amino acids of nectin-1 mutated in this study. The linear epitopes for anti-nectin-1 MAbs CK6 and CK8 were mapped to a region indicated by double underlining (18), and the region of nectin-1 identified as sufficient to confer HSV entry activity to CD155 is indicated by a broken line (8). The sequences shown are those in GenBank entries for nectin-1 (accession number AF060231) and nectin-2 (accession number AF058448).

FIG. 2.

Cell surface expression of nectin-1 mutants. CHO-K1 cells were transfected with plasmids expressing nectin-1 mutants or wild-type nectin-1 or with a control plasmid. After 48 h, the cells were reacted with anti-nectin-1 MAbs and fixed. Binding was detected with a biotinylated secondary antibody followed by streptavidin-horseradish peroxidase. The results are expressed as optical densities at 370 nm (OD₃₇₀) normalized to the binding obtained with wild-type nectin-1 (set to 100% and represented in the graph by a broken line). Values shown are the means and standard deviations of triplicate determinations. Vertical lines dividing the data represent separate experiments. The OD₃₇₀ for binding of the antibodies to wild-type nectin-1 ranged from 1.25 to 2.5. CK6 and CK8 recognize linear epitopes, and R1.302 recognizes a conformational epitope, all in the V domain.

FIG. 3.

HSV entry activities of wild-type nectin-1 and nectin-1 mutants. CHO-K1 cells transfected with plasmids expressing wild-type nectin-1 or nectin-1 mutants or with a control plasmid were replated on 96-well plates (2 × 10⁴ to 4 × 10⁴ cells per well) and exposed to serial dilutions of β-Gal reporter viruses (HSV-1, HSV-1/Rid1, and HSV-2) for 6 h. Then, the β-Gal substrate ONPG was added, and the reaction products were quantitated with a spectrophotometer. Values shown (optical density at 410 nm) are the means and standard deviations for triplicate samples. Similar results were obtained in two other experiments. a.a., amino acid.

FIG. 4.

HSV-1 entry activities of wild-type nectin-1 and nectin-1 mutants at a single input concentration of HSV. Data from Fig. 3 are presented to show the relative entry activities of wild-type and mutant forms of nectin-1 after exposure of the transfected cells to HSV-1 at a single input concentration of 3 × 10⁵ PFU/well. The values obtained with the nectin-1 mutants were normalized to the value obtained with wild-type nectin-1, which was set to 100% and which is represented in the graph by a broken line. Standard deviations of triplicate determinations for each sample are shown.

FIG. 5.

Entry activities of wild-type nectin-1 and nectin-1 mutants for PRV and BHV-1. CHO-K1 cells transfected with plasmids expressing wild-type nectin-1 or nectin-1 mutants or with a control plasmid were replated on 96-well plates (2 × 10⁴ to 4 × 10⁴ cells per well) and exposed to serial dilutions of β-Gal reporter viruses (PRV and BHV-1) for 6 h. Then, the β-Gal substrate ONPG was added, and the reaction products were quantitated with a spectrophotometer. A representative experiment is shown. Values shown (optical density at 410 nm) are the means and standard deviations for triplicate samples.

FIG. 6.

Binding of soluble gD molecules to wild-type nectin-1 and nectin-1 mutants. CHO-K1 cells transfected with plasmids expressing nectin-1 mutants or wild-type nectin-1 or with a control plasmid were replated on 96-well plates. The cells were incubated with serial dilutions of culture supernatants containing the various gD:Fc molecules, at the concentrations indicated, for 30 min and fixed. Binding was detected with a biotinylated secondary antibody followed by streptavidin-horseradish peroxidase. Values shown (optical density at 380 nm [OD₃₈₀]) are the means and standard deviations of triplicate determinations.

FIG. 7.

Locations of the critical mutations on a model of the V domain of nectin-1. The model presented is based on the model of the poliovirus receptor, CD155, deposited in the Brookhaven Protein Database as entry 1DGI (16). The backbone trace of the V domain is colored from dark blue at the N terminus (N) to green at the C terminus (C), which is connected to the second immunoglobulin-like domain. Red coloring denotes the predicted locations of Met85 and Asn77. Amino acid substitutions at both of these positions significantly reduced entry activities for HSV strains but not for PRV or BHV-1.