Identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein G

Abstract

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.

FIG. 1

RSV G glycoprotein ectodomain consensus sequences. Fifteen-amino-acid long overlapping peptides were generated for both RSV subgroups. Consensus peptides (RSVCONSA and -B) are aligned with subgroup A (strain A2) and subgroup B (strain 18537) viruses to demonstrate homology of the consensus peptides with wild-type virus. Peptides are numbered 1 through 44 (subgroup A) or 1 through 45 (subgroup B) starting from the carboxy terminus. The highlighted sequence represents the exactly conserved region (amino acids 164 to 176), and conserved cysteine residues are marked by black dots. The putative heparin binding region of the RSV-G ectodomain (amino acids 187 to 217) (27), characterized by a cluster of basic amino acids, is defined by the black box. Peptides for subgroup A (solid lines) and subgroup B (dashed lines) indicate the positions of the peptides that bound heparin.

FIG. 2

HAAC of pooled biotinylated peptides. One milliliter of peptide (100 μg/ml) in carbonate-bicarbonate buffer (pH 9.6) was run over heparin agarose columns. The columns were washed with 20 column volumes of carbonate-bicarbonate buffer (pH 9.6) containing 100 mM NaCl and 0.1% Triton X-100 followed by 10 column volumes of carbonate-bicarbonate buffer only. Bound peptides were eluted in carbonate-bicarbonate buffer containing 2 mg of heparin/ml. Eluted biotinylated peptides were adsorbed to microtiter plates, and endpoint titers were determined by ELISA. Values above the dashed line (twice background) are considered positive. The data are from a representative experiment of at least three experiments, with the error bars indicating the standard error of the mean of quadruplicate wells.

FIG. 3

Demonstration of the specificity of the HAAC for RSV and other heparin binding peptides. Peptide pools A3 and B3 (100 μg/ml) and control peptides Vn (positive control) and 3vnm (negative control) (10 μg/ml) were reacted with heparin agarose or unlinked CL4B agarose as described in the legend to Fig. 2. Data are from one of two separate experiments, with error bars indicating the standard error of the mean.

FIG. 4

HAAC of individual biotinylated peptides comprising pool 3. The individual peptides (10 μg/ml) were tested for their abilities to bind heparin agarose. Peptides that specifically bound heparin were eluted and detected as described in the legend to Fig. 2. Values equal to or above the dashed line (twice background) are considered positive. The data are from a representative experiment of at least three experiments, with the error bars indicating the standard error of the mean of quadruplicate wells.

FIG. 5

Reactivities of pooled biotinylated peptides with Vero cells. Subgroup A and B peptide pools (100 μg/ml) were diluted in BLOTTO–0.05% Tween 20 with or without the addition of heparin (2 mg/ml) and tested for their abilities to bind to Vero cells. Bound peptides were detected with avidin conjugated with HRP (1:500) and ABTS as a substrate. Values at or above the dashed line (twice background) are considered positive. The data are from a representative experiment of at least three experiments, with the error bars indicating the standard error of the mean of quadruplicate wells.

FIG. 6

Reactivities of individual biotinylated heparin binding peptides with Vero cells. The individual peptides from pool A3 and B3 shown previously to interact with heparin were tested for their abilities to bind Vero cells in the presence (diagonally hatched bars, subgroup A; crosshatched bars, subgroup B) and absence (open bars, subgroup A; solid bars, subgroup B) of 2 mg of heparin/ml. Individual peptides were tested at a concentration of 10 μg/ml and detected as described in the legend to Fig. 5. Values at or above the dashed line (twice background) are considered positive. The data are from a representative experiment of at least three experiments, with the error bars indicating the standard error of the mean of quadruplicate wells.

FIG. 7

Reactivities of pooled biotinylated peptides with various CHO cell lines. Pooled peptides were reacted with various CHO cells, and after fixation, bound peptides were detected as described in the legend to Fig. 5. Values at or above the dashed line (twice background) are considered positive. The data are from a representative experiment of at least three experiments, with the error bars indicating the standard error of the mean of quadruplicate wells.

FIG. 8

Reactivities of individual biotinylated peptides with various CHO cell lines. Individual peptides from pools A3 and B3 shown previously to interact with heparin were tested for their abilities to bind various CHO cell lines. Individual peptides were tested at a concentration of 10 μg/ml and, after fixation, were detected as described in the legend to Fig. 5. Values at or above the dashed line (twice background) are considered positive. The data are from a representative experiment of at least three experiments, with the error bars indicating the standard error of the mean of quadruplicate wells.

FIG. 9

Inhibition of homologous and heterologous RSV infectivity by heparin binding peptides. Vero cells were preincubated with 50 μM peptide per well in 50 μl. Peptide was allowed to adsorb for 45 min at 37°C, followed by the addition of 100 TCID₅₀ of virus (solid bars, strain A2; open bars, strain 18537) for 2 h. The cells were washed and overlaid with EMEM–1% FBS–1% methylcellulose for 3 days before being fixed and stained with 1% crystal violet. 3vnm was used as a negative control for peptide inhibition of virus infectivity. Infectivity was determined as a percentage versus an untreated control. The error bars represent the standard error of the mean of quadruplicate measurements from three separate experiments. The test peptide results were considered significant, with P values of <0.05 versus control peptides (*).