Soluble Nec1₁₂₃-mediated HSV-1 infection of gD receptor-deficient cells. (A) Comparison of virus entry mediated by soluble nectin-1 V domain and cellular nectin-1. CHO-K1 and CHO-Nec1 cells were infected in suspension with QOZHG at different MOIs in the presence (CHO-K1) or absence (CHO-Nec1) of 450 nM sNec1₁₂₃. The cells were plated, and β-galactosidase expression in triplicate cultures was measured by ONPG assay. (B) Comparison of sNec1₁₂₃-mediated infection of CHO-K1 and J1.1-2 cells. QOZHG and sNec1₁₂₃ (500 nM) were mixed and incubated for 1 h at 4°C prior to infection of CHO-K1 or J1.1-2 cells in suspension at an MOI of 3 for 1 h at 37°C. The cells were then plated, and virus entry was visualized by X-Gal staining 15 h later. Both experiments in this figure (A and B) were repeated twice with similar results.

Soluble Nec1₁₂₃-mediated HSV-1 infection of CHO-K1 cells. CHO-K1 cells in suspension were incubated with reporter virus QOZHG at three different MOIs for 1 h at 4°C and infected for 1.5 h at 37°C in the presence of increasing concentrations of sNec1₁₂₃ protein. The cells were then collected and plated. (A) Monolayers stained with X-Gal 16 h after plating. (B) Enlarged images of stained monolayers were used to count the number of blue and total cells in defined fields, and the results were computed as the percentage of blue cells in each well. (C) Separate monolayers were collected for quantitative measurement of β-galactosidase expression by ONPG assay. Results in panels B and C represent averages of triplicate determinations.

Requirement for GAG binding in sNec1₁₂₃-mediated infection. (A) Heparin inhibition of sNec1₁₂₃-mediated virus entry. Different dilutions of QOZHG were preincubated with or without 25 μg/ml heparin for 1 h at 4°C and incubated with CHO-K1 cells in suspension for 30 min at 4°C prior to the addition of 450 nM sNec1₁₂₃ and infection for 1 h at 37°C. The cells were plated with fresh medium, and entry was measured by ONPG assay the next day. Data represent the averages of triplicate determinations. (B) Heparin inhibition of infection via cellular nectin-1. QOZHG was preincubated with heparin as above and adsorbed to CHO-Nec1 cells. Infection was as described above without added sNec1₁₂₃ protein. The cells were processed for ONPG assay as above. (C) Infection of GAG-deficient CHO cells. CHO-K1 and GAG-deficient pgsD-677 and pgsA-745 cells were infected with QOZHG at an MOI of 3 in the presence (black bars) or absence (gray bars) of 450 nM sNec1₁₂₃ protein. Virus entry was determined by ONPG assay. Values are averages from triplicate infections.

Examination of sNec1₁₂₃ association with the target cells. (A) Effect of the His₆ tag on sNec1₁₂₃-mediated entry. Left, soluble nectin-1 V domain with (+His) or without (ΔHis) His tag was produced by transfection of 293T cells and identified in the culture medium by immunoblotting using nectin-1 (CK6) and His tag-specific antibodies. Concentrations of the recombinant proteins were determined by densitometry comparison to purified sNec1₁₂₃ standards (data not shown). Right, different amounts of the collected 293T medium (expressed as nM recombinant protein) were included in KHZ.1 infections of CHO-K1 cells at an MOI of 3. Entry was determined by ONPG assay. (B) Effects of preincubation of CHO-K1 cells with sNec1₁₂₃ and/or soluble gD ectodomain (sgD₂₈₇) on QOZHG infection (MOI = 1) in the presence (gray bars) or absence (open bars) of 500 nM sNec1₁₂₃. Concentrations of soluble proteins during the preincubation period are indicated at the bottom. Following the preincubation, cells were washed and incubated with virus for 1 h at 4°C prior to infection at 37°C for 1 h in the presence or absence of freshly added sNec1₁₂₃. The cells were collected and plated with fresh medium, and entry was determined by ONPG assay the following day. The two panels show independent experiments, each performed in triplicate.

Specificity of sNec1₁₂₃-mediated entry. Results are from ONPG assays 16 h after plating. (A) Comparison of QOZHG entry mediated by sNec1₁₂₃ and the gD binding-defective mutant derivative sNec1₁₂₃TMC. CHO-K1 cells were infected at an MOI of 1 in the presence of increasing amounts of sNec1₁₂₃ or sNec1₁₂₃TMC protein. (B) Virus entry mediated by soluble nectin-2 V domain (sNec2₁₃₅). CHO-K1 cells were infected with replication-competent, lacZ-expressing viruses containing wild-type gD (KHZ.1) (60) (MOI = 12) or rid1 mutant gD (KOS-Rid1/tk12) (77) (MOI = 6) in the presence of increasing amounts of sNec2₁₃₅ protein. (C) Entry of KHZ.1 (MOI = 6) and KOS-Rid1/tk12 (MOI = 3) in the presence of sNec1₁₂₃. (D) Inhibition of the entry-promoting activity of sNec1₁₂₃ by preincubation of the soluble receptor with soluble gD ectodomain, sgD₂₈₇. Soluble receptor and soluble gD were mixed in the indicated molar proportions (nM/nM) and incubated for 15 min at 4°C. QOZHG was then added, and the mixture was incubated for another 15 min at 4°C. CHO-K1 cells in suspension were then infected at 37°C for 50 min (MOI = 3) and plated with fresh medium. The numbers under the bars show the concentrations of the two soluble molecules during infection. Results are expressed as percent infection relative to the positive (360/0) control. (E) Inhibition of infection of receptor-bearing cells by soluble gD. CHO-Nec1 cells in suspension were incubated with sgD₂₈₇ at the indicated concentrations for 30 min at 4°C prior to infection with QOZHG (MOI = 3) for 50 min at 37°C. Results are expressed as percent infection relative to the control without sgD. All results represent averages of triplicate determinations, and each experiment was performed at least twice.