Human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment - PubMed (original) (raw)

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

Markete Vlasak et al. J Virol. 2005 May.

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.

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Figures

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.

References

1. Baranowski, E., C. M. Ruiz-Jarabo, and E. Domingo. 2001. Evolution of cell recognition by viruses. Science 292:1102-1105. - PubMed
1. Bayer, N., E. Prchla, M. Schwab, D. Blaas, and R. Fuchs. 1999. Human rhinovirus HRV14 uncoats from early endosomes in the presence of bafilomycin. FEBS Lett. 463:175-178. - PubMed
1. Bayer, N., D. Schober, E. Prchla, R. F. Murphy, D. Blaas, and R. Fuchs. 1998. Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection. J. Virol. 72:9645-9655. - PMC - PubMed
1. Bernard, K. A., W. B. Klimstra, and R. E. Johnston. 2000. Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice. Virology 276:93-103. - PubMed
1. Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu. Rev. Cell Biol. 8:365-393. - PubMed

Human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment - PubMed (original) (raw)