Human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment

Abstract

We have previously isolated mutants of the major-group human rhinovirus type 89 that grow in cells deficient in intercellular adhesion molecule 1 (ICAM-1), the receptor used by the wild-type virus for cell entry [A. Reischl, M. Reithmayer, G. Winsauer, R. Moser, I. Goesler, and D. Blaas., J. Virol. 75:9312-9319, 2001]. We now demonstrate that one of these variants utilizes heparan sulfate proteoglycan (HSPG) as a cellular receptor. Adaptation to ICAM-1-deficient cells not only resulted in the newly acquired receptor specificity but also rendered the virus less stable at low pH and at elevated temperatures. This instability might compensate for the absence of the uncoating activity of ICAM-1. Whereas wild-type virus infection via ICAM-1 proceeded in the presence of the vesicular H(+)-ATPase inhibitor bafilomycin A1, infection by the mutant via HSPG was prevented by the drug. This suggests that the low pH prevailing in endosomal compartments is required for uncoating in the absence of the catalytic activity of ICAM-1.

FIG. 1.

Binding of HRV89₁₅ to COS-7 cells is inhibited by sulfated glycans. COS-7 cells grown in 24-well plates were incubated for 1 h at 37°C with radiolabeled HRV89₁₅ in the presence of 2 mg/ml of the proteoglycans indicated (DS, dermatan sulfate; CS, chondroitin sulfate). The supernatant was removed, the cells were washed, and cell-associated radioactivity versus total radioactivity was determined by liquid scintillation counting. Since the absolute values obtained differed somewhat from experiment to experiment, virus bound in plain medium (control) was set to 100% in each experiment and the other values were normalized accordingly. The mean ± standard deviation of three experiments carried out in parallel is shown.

FIG. 2.

Binding of HRV89₁₅ is diminished upon treatment of COS-7 cells with enzymes degrading proteoglycans. COS-7 cells grown in 24-well plates were incubated for 2 h at 37°C with 0.2 U/ml neuraminidase and 2 U/ml other enzymes in PBS⁺⁺ as indicated. The cells were washed with cold PBS, and 10⁵ TCID₅₀ of HRV89₁₅ in 200 μl cold medium was added. After incubation for 1 h at 4°C, the supernatant was removed, the cells were washed with cold PBS, 200 μl infection medium was added, and plasma membrane bound virus was released by three freeze-thaw cycles. Cell debris was removed by low-speed centrifugation, and virus present in the supernatant was determined by end point dilution and is given as a percentage of the control (incubation in PBS without any addition). The mean ± standard deviation of three independent experiments is shown.

FIG. 3.

Growth of COS-7 cells in the presence of chlorate reduces viral binding. Cells were grown for 3 days in medium containing 50 mM NaClO₃, washed, incubated with radiolabeled HRV89₁₅ for 1 h, and washed again, and cell-associated radioactivity was determined by liquid scintillation counting. Values are given as percentages of virus binding to cells grown in the absence of chlorate (control). The mean ± standard deviation of three experiments carried out in parallel is shown.

FIG. 4.

Binding of HRV89₁₅ to cells deficient in HSPG synthesis is reduced. CHO-K1 (K1), wt; pgsA-745 (A 745), deficient in xylosyltransferase; pgsD-677 (D 677), doubly deficient in N-acetylglucosaminyltransferase and glucuronyltransferase. Cells were grown in 24-well plates and challenged with about 20,000 cpm of virus for 60 min at 4°C. Cell-associated radioactivity was determined by scintillation counting. Virus bound to the cells is expressed as a percentage of total radioactivity added. Wild-type HRV89 was included as a control. The mean ± standard deviation of three experiments carried out in parallel is shown.

FIG. 5.

Thermal inactivation of HRV89₁₅ compared to wt HRV89. Virus was incubated at 47°C in PBS, and viral infectivity was determined by end point dilution assays at the indicated time points. The mean of three independent experiments ± standard deviation is shown.

FIG. 6.

Sensitivity of mutant HRV89 to low pH compared to wt HRV89. Virus was incubated in Na⁺-acetate buffer of pH 4.5 for the times indicated at room temperature, and residual viral infectivity was determined by end point dilution assay. The mean ± standard deviation of three independent experiments is shown.

FIG. 7.

HRV89₁₅ is dependent on low endosomal pH for infection. COS-7 cells (A) and HeLa cells (B) grown in 24-well plates were preincubated with and without 200 nM bafilomycin (baf) A1 for 60 min at 34°C as indicated, challenged with HRV89₁₅, and further incubated at 34°C in the presence of 100 nM of the drug. At the times indicated, the plates were subjected to three freeze-thaw cycles, cell debris was removed by low-speed centrifugation, and the viral titer in the supernatant was determined. Where indicated, bafilomycin and/or the ICAM-1 blocking antibody R6.5 was present during the whole experiment. The mean of four independent experiments ± standard deviation is shown.

FIG. 8.

Low concentrations of heparin stimulate infection. COS-7 cells grown in 96-well plates were challenged with HRV89₁₅ at 10⁷ TCID₅₀/well in the presence of the indicated concentrations of heparin. After incubation at 34°C for the times indicated, cell viability was assessed. Control, noninfected cells (n.i.). Viability was determined in a microplate reader at 495 nm. A typical experiment out of four with the mean of four parallel determinations ± standard deviation is shown.