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Biochemical characterization of a glycoprotein required for rhinovirus attachment
J E Tomassini1, T R Maxson, R J Colonno
1Department of Virus and Cell Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
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.
Insights
Human rhinoviruses use a 90-kDa cell surface protein for host cell attachment. While carbohydrates, including sialic acid, are present, they are not the primary component of the receptor binding site.
Area of Science:
- Virology
- Cell Biology
- Glycobiology
Background:
- Human rhinoviruses initiate infection by attaching to host cell receptors.
- A 90-kDa cell surface protein is known to mediate human rhinovirus attachment.
- The precise nature of the receptor's interaction with the virus requires further elucidation.
Purpose of the Study:
- To characterize the carbohydrate composition of the human rhinovirus receptor.
- To determine the role of receptor carbohydrates in human rhinovirus attachment.
- To investigate the contribution of sialic acid to virus-receptor interactions.
Main Methods:
- Enzymatic digestion of the purified receptor protein with glycosidases.
- Wheat germ lectin inhibition assays to assess carbohydrate involvement.
- Tunicamycin treatment and N-glycanase digestion to identify N-linked glycosylation.
- Isoelectric focusing to compare native and deglycosylated receptor properties.
- Analysis of virus and antibody binding to neuraminidase-treated host cell membranes.
Main Results:
- Approximately 30% of the receptor's molecular mass consists of complex-type oligosaccharides, with sialic acid contributing one-third.
- Wheat germ lectin inhibited rhinovirus attachment, indicating sialic acid's role.
- Oligosaccharides were confirmed as N-linked, with seven N-linked glycosylation sites detected.
- Deglycosylation shifted the receptor's isoelectric point from 4.2 to 5.3.
- Carbohydrates are involved but not the predominant component of the receptor binding site.
Conclusions:
- The human rhinovirus receptor is a glycoprotein with significant N-linked glycosylation.
- Sialic acid residues on the receptor play a role in, but are not solely responsible for, virus attachment.
- The primary binding interaction between human rhinoviruses and their receptor likely involves non-carbohydrate components.