Receptor-binding properties of a soluble form of human cytomegalovirus glycoprotein B

Abstract

The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (Kd): Kd1, 4.96 x 10(-6) M; Kd2, 3.07 x 10(-7) M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (Kd1, 9.85 x 10(-6) M; Kd2, 4.03 x 10(-8) M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (Kd, 7.46 x 10(-6) M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.

FIG. 1

Purity of [³⁵S]gB-S. Eluates from the Ni²⁺-NTA column were subjected to SDS-PAGE analysis under nonreducing conditions on a 7.5% acrylamide gel. Lane 1 shows a [³⁵S]gB-S sample that was electrophoresed, transferred to nitrocellulose, probed with an anti-gB monoclonal antibody (27-78), and detected with a secondary goat anti-mouse HRP-conjugated antibody in conjunction with a chemiluminescent substrate. Lane 2 shows a [³⁵S]gB-S sample that was electrophoresed, after which the gel was dried, fixed, and exposed to film.

FIG. 2

Time course analysis. IF cells were cultured in a 96-well plate and chilled to 4°C, and nonspecific binding sites were blocked for 30 min at 4°C by the addition of 5 mM ovalbumin in PBS-GC. A constant concentration of [³⁵S]gB-S (0.5 mg/ml) was incubated with the cell monolayer for increasing increments of time. At the conclusion of the designated time, unbound [³⁵S]gB-S was removed, the cells were washed twice with PBS-GC, and the cells containing the bound gB-S were lysed by the addition of 1% SDS–1% Triton X-100. Experiments for all data points were performed in triplicate. The specific activity of the [³⁵S]gB-S preparation used for this experiment was 153.55 cpm/μg of protein.

FIG. 3

Dose response binding curve. IF cells cultured in a 24-well plate were chilled and blocked at 4°C. Increasing concentrations of [³⁵S]gB-S, diluted in PBS-GC, were added to the cell monolayers and incubated for 90 min at 4°C. Unbound and bound fractions were collected and subjected to scintillation counting. Experiments for all data points were performed in duplicate. Bars represent standard deviations. The specific activity of the [³⁵S]gB-S preparation used for this experiment was 267 cpm/μg of protein. On the basis of the specific activity of the [³⁵S]gB-S preparation, the amounts of total input and free and bound proteins were determined and subjected to Scatchard analysis (49). Shown in the inset are lines representing the best fits as determined by a linear regression analysis. The experiment was repeated several times with consistent results.

FIG. 4

Homologous competition assay. A constant concentration of [³⁵S]gB-S (0.2 mg/ml) was added to IF cells in the presence of increasing concentrations of nonradiolabeled gB-S for 90 min at 4°C. Each data point represents the average of duplicate wells. Bars represent standard deviations. The specific activity of the [³⁵S]gB-S preparation used for this experiment was 230 cpm/μg of protein. Nonspecific binding was determined by the addition of a 100-fold molar excess of nonradiolabeled gB-S and was determined to be 21%. Nonspecific binding was deducted from each data point prior to graphing.

FIG. 5

Binding of [³⁵S]gB-S in the absence of HSPGs. IF cell monolayers were treated with increasing concentrations of heparinase (□) or chondroitinase ABC (⧫) for 60 min at 37°C. Binding assays were conducted for 90 min with 0.5 mg of [³⁵S]gB-S per ml diluted in PBS-GC. Each data point represents the average of duplicate wells. Bars represent standard deviations. The specific activity of the [³⁵S]gB-S preparation used for this experiment was 368.05 cpm/μg of protein.

FIG. 6

Saturable binding of gB-S to CHO cell monolayers. CHO-K1 (□) and CHO-677 (⧫) cells were cultured in a 96-well plate, chilled, and blocked at 4°C. Increasing concentrations of [³⁵S]gB-S were added to the cell monolayers, which were incubated for 90 min at 4°C. Unbound and bound fractions were collected and subjected to scintillation counting. Experiments for all data points were performed in triplicate. Bars represent standard deviations. The specific activity of the [³⁵S]gB-S preparation used for this experiment was 320.5 cpm/μg of protein. On the basis of the specific activity of the [³⁵S]gB-S preparation, the amounts of total input and free and bound proteins were determined and subjected to Scatchard analysis (49). Shown in the inset are lines representing the best fits as determined by a linear regression analysis.

FIG. 7

HCMV infection is decreased in the presence of gB-S. IF monolayers cultured on glass coverslips in a 12-well plate were chilled to 4°C. Increasing concentrations of nonradiolabeled gB-S (□), BSA (⧫), or 10 μg of heparin per ml were allowed to incubate with the cells for 60 min at 4°C. Virus was added to the cells (MOI = 0.1) and allowed to absorb for 90 min at 4°C. After a 30-min temperature shift to 37°C, a low-pH citrate buffer was added to inactivate any extracellular virus. At 24 h postinfection, immunofluorescence staining was performed with a rabbit anti-IE antibody followed by the secondary goat anti-rabbit–fluorescein conjugate in combination with Hoechst dye. Experiments for all data points were performed in duplicate, and a minimum of 500 cells per coverslip were scored. Bars represent standard deviations.

FIG. 8

Soluble form of gB blocks HCMV attachment. IF cells were treated with increasing concentrations of gB-S or 10 μg of heparin per ml for 60 min, after which [³⁵S]methionine gradient-purified HCMV virions were added for 90 min. All samples were tested in triplicate.