Abstract
Human rhinoviruses attach to specific receptors located on the surfaces of host cells as a first step in viral infection. A 90-kDa cell surface protein was previously shown to be involved in the attachment of human rhinoviruses to susceptible cells (Tomassini, J. E., and Colonno, R.J. (1986) J. Virol. 58, 290-295). Digestion of purified receptor protein with various glycosidases revealed that 30% of its molecular mass was comprised of complex-type oligosaccharides, one-third being contributed by sialic acid. The presence of sialic acid was confirmed by demonstrating that wheat germ lectin can inhibit the attachment of rhinoviruses to host cell membranes, while lectins of other sugar specificities had no effect. The oligosaccharides were shown to be N-linked by tunicamycin treatment of host cells and by N-glycanase digestion. Seven N-linked glycosylation sites were detected by partial digestion of the receptor oligosaccharides with N-glycanase. Native receptor protein had an isoelectric focusing point of 4.2, compared to 5.3 for the deglycosylated protein. Studies of virus and antibody binding to neuraminidase-treated host cell membranes suggested that although carbohydrates may be involved in host-virus interaction, the receptor carbohydrate is not the predominant component of the cellular receptor site.
Highlights
Human rhinoviruses attach to specific receptors lo- examining the effect on virus binding
Exoglycosidase Digestion of Human rhinoviruses (HRVs) Receptor Protein-A cell protein was iodinated by the chloramine-T procedure [20] and repu- surface protein required for attachment of the vast majority rified by gel filtration prior to subsequent analysis
A previous study reported the isolation of a receptor protein involved in the attachment of the majority of HRVs to host cells [19]
Summary
Vs of radioactivity in the supernatants was determined. Gels were fixed and stained by the method of Merrill et al [26]. Chased from Calbiochem; 0- and N-glycanases were from Genzyme; ReceptorIsolation-To prepare adequate amounts of the HRV endoglycosidasesD, H, and F, from Boehringer Mannheim; P-galacreceptor protein for subsequent biochemical characterization, 40-50 tosidase, cy- and @glucosidases, and a-mannosidase, from Sigma. 0.3% sodium deoxycholate,passed over an anti-bovine serum albumin possible under conditions as described above. Thenpurified by affinity chromatography using coupled from Sigma. 50 pg of purified receptor protein could be routinely recovered following extensive washing of the columns with 0.5% sodium deoxycholate in 20 mM Tris-HC1, pH 7.4, and elution with 50 mM diethylamine, pH 11.5.
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