cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1.

Abstract

A 90-kDa surface glycoprotein was previously isolated and shown to be required for infection by the "major" group of human rhinovirus (IRV) serotypes. In the present work, the amino acid sequence of the receptor protein was obtained from CNBr and tryptic peptides. Using degenerate oligonucleotides predicted from the peptide sequences, we identified four cDNA clones that encode a 3-kilobase mRNA. The clones were ligated, subcloned in a simian virus 40 expression vector, and used to transfect receptor-negative Vero (monkey) cells. Results showed that transfected cells expressed receptor molecules capable of binding HRV and a monoclonal antibody which recognizes the mjor group HRV receptor. The cloned receptor cDNA encoded a protein with a sequence nearly identical to that of the intercellular adhesion molecule 1 (ICAM-1), indicating that the two surface proteins are one and the same. Both proteins have identical mass, carbohydrate composition, and tissue distribution. In addition, major group receptors on HeLa cells could be induced with various cytokines in a manner similar to the ICAM-1 ligand. A similar induction of the HRV "minor" group receptor was not observed.Human rhinoviruses (HRVs) are members of the Picornaviridae and are the major causative agent of the common cold in humans (1). Earlier studies have indicated that HRV serotypes can be divided into "major" (78 serotypes) and "minor" (10 serotypes) groups based on receptor specificity (2,3). An anti-receptor monoclonal antibody (designated antibody 1A6) has been previously isolated which specifically blocks attachment of the major group of HRVs (4). By using this antibody, a 90-kDa glycoprotein was isolated from HeLa cell membranes and shown to be required for attachment of the major group of HRVs to susceptible cells in culture (5). Chimpanzee and human clinical trials testing the efficacy of antibody 1A6 have also demonstrated that this receptor is involved in HRV infection of the nasal cavity in vivo (6, 7). Further biochemical characterization of the purified 90-kDa protein demonstrated that it was an acidic glycoprotein having a pI of4.2 (8). Carbohydrates accounted for 30% ofthe molecular mass of the protein and seven N-glycosylation sites were predicted on the basis of partial digestion with N-Glycanase (8).The intercellular adhesion molecule 1 (ICAM-1) is a cell surface ligand for the lymphocyte function-associated antigen 1 (LFA-1) adhesion receptor (9). ICAM-1 is a singlechain 76-to 114-kDa glycoprotein with a polypeptide core of 55 kDa that can be induced by several cytokines (10). The interaction of ICAM-1 and LFA-1 plays an important role in leukocyte adhesion and in the execution of immunological and inflammatory functions (10).The present study describes the cloning and sequencing of the cDNA that encodes the cell surface receptor required for infection by the major group ofHRVs.t Sequence and protein comparisons show that ICAM-1 is the surface glycoprotein utilized by HRVs during infection. MATERIALS AND METHODSPeptide Sequence....

Inhibition of Clathrin-dependent Endocytosis Has Multiple Effects on Human Rhinovirus Serotype 2 Cell Entry

“…They were then incubated in fresh medium containing 50 g/ml FITC-transferrin for 20 min at 37°C and cooled. Plasma membrane-bound transferrin was removed by acid wash (see above), followed by a wash with PBS, fixed with 4% paraformaldehyde for 1 h at room temperature, quenched with 50 mM NH 4 Cl, permeabilized with 0.05% saponin, and incubated with rhodamine-labeled anti-HA monoclonal antibody (1:80 dilution). For HRV2 internalization, cells were then incubated with HRV2 at a m.o.i.…”

“…Cells were then transferred to 4°C and washed with ice-cold PBS 2ϩ , and plasma membrane-bound virus was masked by incubating the cells with rabbit anti-HRV2 antiserum (preadsorbed with non-infected cells and diluted 1:10) for 1 h at 4°C. Cells were washed with PBS 2ϩ , fixed with 4% paraformaldehyde in PBS for 1 h at room temperature, quenched with 50 mM NH 4 Cl, and permeabilized with 0.05% saponin. First, internalized D-and C-antigenic HRV2 was detected by indirect immunofluorescence using mAb 8F5 at 6 g/ml and Alexa 488-labeled goat anti-mouse antibody (1:500).…”

“…These small RNA-containing viruses constitute a large genus within the family Picornaviridae. The Ͼ100 serotypes are divided into two groups based on their receptor specificity (1, 2); major group viruses (91 serotypes) have access to the host cell by binding to intercellular adhesion molecule-1 (ICAM-1/CD54) 1 (3)(4)(5), whereas minor group viruses (10 serotypes) bind to members of the low density lipoprotein receptor (LDLR) family (6,7). Internalization into bona fide endosomes has been demonstrated for the minor group virus HRV2 and the major group virus HRV14 (8).…”

Soluble receptors in human disease

“…Elevated soluble ICAM-1 levels are found in patients with congenital leukocyte adhesion deficiency and may contribute to that disease [94]. ICAM-1 is also the receptor for human rhinovirus, a principal cause of seasonal upper respiratory infections [95,181,182]. A soluble ICAM-1 derived by genetic truncation prevents viral infection by direct competition with viral binding sites [183].…”

The Minor Receptor Group of Human Rhinovirus (HRV) Includes HRV23 and HRV25, but the Presence of a Lysine in the VP1 HI Loop Is Not Sufficient for Receptor Binding

“…Later, based on competition for cellular binding sites, two different groups of viruses using nonidentical receptors for cell attachment were defined within the genus Rhinovirus (18). Subsequently, 24 (1) and finally 100 serotypes (counting the subtypes HRV1A and HRV1B as one strain) were assigned to the two receptor groups by using cross-competition and inhibition of cell binding by a monoclonal antibody recognizing intercellular adhesion molecule 1 (ICAM-1), the receptor of the major group of HRVs (32,33,35). According to these reports, 90 serotypes bind ICAM-1, whereas both subtypes of HRV1 (HRV1A and HRV1B) and 8 other serotypes were categorized as belonging to the minor group; they were later shown to use members of the low-density-lipoprotein receptor (LDLR) family for cell entry (12,20,36).…”