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. 2000 Jul;74(14):6442-7.
doi: 10.1128/jvi.74.14.6442-6447.2000.

The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate

S A Feldman  1 , S Audet, J A Beeler
Affiliations

The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate

S A Feldman et al. J Virol. 2000 Jul.

Abstract

Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED(50)] = 0.28 +/- 0.11 microg/ml) and cp-52 (ED(50) = 0.55 +/- 0. 14 microg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.

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Figures

FIG. 1
FIG. 1
Heparin-agarose affinity chromatography of RSV (strain A2)-infected Vero cell lysates. HAAC was carried out on infected cell lysates (250 μg of protein per ml of heparin-agarose) followed by immunoprecipitation. Samples were analyzed by SDS-PAGE (4 to 20% gels) followed by Western blot with a polyclonal rabbit anti-G (A) or anti-F (B) antiserum. The infected cell lysate was analyzed for the presence of RSV-specific protein (lane 1) prior to being run over heparin-agarose columns. Columns were washed twice with 10 column volumes of MES buffer, and the final wash was examined (lane 2). Bound proteins were eluted with MES buffer containing heparin (2 mg/ml) (lane 3). As a control to demonstrate that the viral proteins were specifically binding to heparin, identical samples were run over columns containing uncoupled CL-4B agarose, and the heparin-eluted material was examined for the presence of RSV-G and -F proteins (lane 4). In addition, uninfected cell lysate was run over heparin-agarose to control for polyclonal antibody reactivity (lane 5). G90 represents the mature, fully glycosylated form of RSV-G. G55 is a partially glycosylated precursor of G90.
FIG. 2
FIG. 2
Heparin-agarose affinity chromatography of RSV-F proteins. Purified RSV proteins or infected cell lysates from recombinant vaccinia viruses expressing either RSV-G (Ga, subgroup A; Gb, subgroup B) or RSV-F were subject to HAAC followed by Western blot analysis using a polyclonal rabbit anti-G (A) or anti-F (B) antiserum. The assay was carried out as described in Materials and Methods except that purified proteins were used at a concentration of 50 μg/ml of heparin-agarose. (A) Purified RSV-Ga (lane 1), vaccinia virus-expressed RSV-Ga (lane 2), and vaccinia virus-expressed RSV-Gb (lane 3). (B) Purified RSV-Fa (lane 1) and vaccinia virus-expressed RSV-Fa (lane 2). G90 represents the mature, fully glycosylated form of RSV-G.
FIG. 3
FIG. 3
Heparin-agarose affinity chromatography of RSV cp-52-infected Vero cell lysate. Heparin chromatography was performed as described in the legend to Fig. 1, followed by Western blot analysis using an anti-F polyclonal antiserum (lane 1, infected cell lysate; lane 2, final wash; lane 3, heparin-eluted material). To demonstrate that cp-52 is unable to synthesize the G and SH proteins, cp-52 lysate was probed with an anti-G polyclonal antiserum (lane 4) or an anti-SH antiserum (lane 6), and lysate from A2-infected cells was included as a control for anti-G (lane 5) and anti-SH (lane 7) antibody reactivity. G90 represents the mature, fully glycosylated form of RSV-G. G55 is a partially glycosylated precursor of G90. SH0 represents the full-length unglycosylated form of RSV-SH.
FIG. 4
FIG. 4
Inhibition of virus infectivity by various GAGs. RSV A2 or cp-52 or measles virus (100 TCID50) was incubated with BVL heparin (●), LMW heparin (□), de-N-sulfated heparin (■), or chondroitin sulfate (○) for 1.5 h at 37°C before being added to cells. The infection was allowed to progress for 72 h (A2 and measles virus), after which the cells were fixed and subjected to RSV- or measles virus-specific ELISA. Absorbance values at 405 nm were converted to percentages, and infectivity was then plotted as percentage of that of a mock-treated control. Due to poor detection by ELISA, cp-52 infections were stopped at 6 days postinfection and the cells were fixed and stained with 1% crystal violet. Plaques were counted, and percentages were determined versus a mock-treated control. Representative data are from separate experiments and represent the mean ± 1 standard error of the mean. For A2 and cp-52, the data are from single experiments of at least four separate experiments with similar results. Measles virus inhibition was conducted in parallel with A2 and cp-52, and data represent a single experiment from at least two separate experiments with similar results.
FIG. 5
FIG. 5
Effect of GAG lyase treatment of Vero cells on RSV cp-52 infectivity. Vero cells were treated for 1 h at 37°C with heparin lyase I (●), heparin lyase III (○), or chondroitinase ABC (□) at the indicated concentrations. Following enzyme treatment, the cells were washed and 100 TCID50 of RSV cp-52 was added for 1 h at 37°C. The inoculum was then removed, and the cells were overlaid with EMEM containing 1% methylcellulose. After 6 days, the cells were fixed and stained with 1% crystal violet. Plaques were counted, and percentages were determined versus a mock-treated control. Data represent the mean ± 1 standard error of the mean of at least three separate experiments. ★, P < 0.05.
FIG. 6
FIG. 6
Competitive binding of RSV to Vero cells. A2 (solid bars) or cp-52 (open bars) was incubated with the indicated concentration of BVL heparin (50 μl total volume) for 30 min at 37°C. Subsequently, 4 × 105 Vero cells (50 μl total volume) were added to each reaction and incubated at 4°C for 1 h with mixing. Cells were washed twice with cold PBS (pH 7.4). The percentage of bound virus was determined versus untreated controls (PBS only). The maximum amount of virus bound in any given assay ranged from 15 to 20% of the total virus input. Each point represents the mean ± 1 standard error of the mean of at least three separate experiments. ∗, P < 0.05.
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