The soluble form of human respiratory syncytial virus attachment protein differs from the membrane-bound form in its oligomeric state but is still capable of binding to cell surface proteoglycans

Abstract

The soluble (Gs) and membrane-bound (Gm) forms of human respiratory syncytial virus (HRSV) attachment protein were purified by immunoaffinity chromatography from cultures of HEp-2 cells infected with vaccinia virus recombinants expressing either protein. Sucrose gradient centrifugation indicated that Gs, which is secreted into the culture medium, remains monomeric, whereas Gm is an oligomer, probably a homotetramer. Nevertheless, Gs was capable of binding to the surface of cells in vitro, as assessed by a flow cytometry-based binding assay. The attachment of Gs to cells was inhibited by previous heparinase treatment of living cells, and Gs did not bind to CHO cell mutants defective in proteoglycan biosynthesis. Thus, Gs, as previously reported for the G protein of intact virions, binds to glycosaminoglycans presented at the cell surface as proteoglycans. Deletion of a previously reported heparin binding domain from Gs protein substantially inhibited its ability to bind to cells, but the remaining level of binding was still sensitive to heparinase treatment, suggesting that other regions of the Gs molecule may contribute to attachment to proteoglycans. The significance of these results for HRSV infection is discussed.

FIG. 1.

SDS-PAGE and Western blotting of Gs and Gm proteins. A scheme of the Gm protein is presented in the upper part of the figure, denoting the hydrophobic region (), the potential N- (▾) and O-glycosylation sites (|), and the cysteine residues (•). Also indicated are the two mucin-like regions. Formation of the Gs form occurs by translation initiation (i) at Met48 and subsequent cleavage (ii) after residue 65. HRSV Gs (lanes 1) and Gm (lanes 2) proteins were purified by immunoaffinity chromatography, as described in Materials and Methods, and analyzed by Coomassie staining of an SDS-PAGE gel (A) and by Western blotting with monoclonal antibody 63G (B). Molecular weight markers are shown on the left. G* indicates low-molecular-weight bands reacting with antibody 63G.

FIG. 2.

Sedimentation of Gs and Gm proteins in sucrose gradients. The purified Gs (A and C) and Gm (B and D) proteins from Fig. 1 were loaded onto preformed 5 to 25% sucrose gradients and then centrifuged and processed as indicated under Materials and Methods. Sedimentation proceeds from left to right. Both samples and gradients shown in panels C and D contained 0.1% octyl-glucoside in PBS, whereas those displayed in panels A and B did not contain detergent. Thirty microliters from each gradient fraction (shown at the top of each panel) were analyzed by Western blotting using antibody 63G. Fractions 5 to 9 of the gradient shown in panel D were pooled together, equilibrated in PBS containing 0.1% octyl-glucoside, and centrifuged a second time in a new 5 to 25% sucrose gradient prepared in PBS containing 0.1% octyl-glucoside. Western blot analysis of fractions obtained from the latter gradient is shown in the inset of panel D. Gradients run in parallel in the absence of detergent were loaded with either BSA (68 kDa), mouse immunoglobulin G1 (IgG1; 150 kDa), or catalase (250 kDa). Fractions of these gradients were analyzed by SDS-PAGE and Coomassie blue staining. The peak of the protein sedimentation profile in each of these gradients is indicated (arrows).

FIG. 3.

Binding of Gs protein to HEp-2 cells. (A) The amounts indicated in each lane of either purified HRSV or Gs protein were analyzed by Western blotting with antibody 63G (12). (B) Corresponding amounts of HRSV (Gm) or Gs, as determined by densitometry of the Western blots shown in panel A, were incubated with HEp-2 cells. Following incubation, the binding of virus and Gs to cells was determined by flow cytometry (shaded histograms), as indicated in Materials and Methods. Negative controls of cells incubated without virus or without Gs are shown in each case (unshaded histograms). (C) The amounts of Gs and Gm used in the flow cytometry assay were converted to arbitrary units and plotted against the mean fluorescent intensity (with background fluorescence subtracted) of the histograms shown in panel B.

FIG. 4.

Binding of Gs to proteoglycan-deficient CHO cells and effect of heparinase treatment on Gs attachment. (A) Twenty microliters of purified Gs (shown in Fig. 1) was incubated with wild-type CHO K1 cells or with the proteoglycan-deficient cell mutants pgsA-745 or pgsD-677. The attachment of Gs to each cell type was measured by flow cytometry, as indicated in Materials and Methods (shaded histograms). The unshaded histograms correspond to cells incubated without Gs. (B) HEp-2 cells (3 × 10⁵) were incubated for 1 h at 37°C with the indicated amounts of heparinase I before 20 μl of Gs was added to assess attachment by flow cytometry. The results are expressed as the percentage of mean fluorescence intensity relative to that of heparinase mock-treated cells after subtraction of background values (without added Gs).

FIG. 5.

Binding of Gs lacking the HBD to HEp-2 cells. Partial sequences of the Gs wild-type and GsΔHBD proteins are shown at the top, denoting the proposed HBD (framed) and the amino acids deleted in the latter protein. (A) Western blot analysis (using antibody 63G) of 20 μl of wild-type Gs and mutant GsΔHBD that were concentrated from the supernatants of HEp-2 cells infected with vaccinia virus recombinants expressing the corresponding proteins. (B) The indicated amounts of each protein were assessed for their ability to bind to HEp-2 cells by flow cytometry (shaded histograms). The unshaded histogram is a negative control representing cells incubated without Gs. (C) HEp-2 cells were either incubated with 80 mIU of heparinase I/ml or mock digested as indicated in Fig. 4 before being incubated with 30 μl of Gs (black) or GsΔHBD (grey) and subjected to flow cytometry to assess attachment. The results are expressed as the percentage of mean fluorescence obtained relative to that produced by the binding of Gs to untreated HEp-2 cells after subtraction of background values (without added Gs).