Human-murine chimeras of ICAM-1 identify amino acid residues critical for rhinovirus and antibody binding.
Human ICAM-1 is the cellular receptor for the major group of human rhinoviruses (HRVs). Previous studies have suggested that the N-terminal domain of ICAM-1 is critical for binding of the major group rhinoviruses. To further define the residues within domain 1 that are involved in virus binding, we constructed an extensive series of ICAM-1 cDNAs containing single and multiple amino acid residue substitutions. In each case, substitutions involved replacement of the human amino acids with those found in murine ICAM-1 to minimize conformational effects. To facilitate the mutagenesis process, a synthetic gene encompassing the first two domains of ICAM-1 was constructed which incorporated 27 additional restriction sites to allow mutagenesis by oligonucleotide replacement. Each of the new constructs was placed into a Rous sarcoma virus vector and expressed in primary chicken embryo fibroblast cells. Binding assays were performed with six major group HRVs, including one high-affinity binding mutant of HRV-14, and two monoclonal antibodies. Results indicated that different serotypes displayed a range of sensitivities to various amino acid substitutions. Amino acid residues of ICAM-1 showing the greatest effect on virus and antibody binding included Pro-28, Lys-29, Leu-30, Leu-37, Lys-40, Ser-67, and Pro-70.
Register
R B
RB
Department of Virus and Cell Biology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Uncapher
C R
CR
Naylor
A M
AM
Lineberger
D W
DW
Colonno
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D002678:Chimera; D000939:Epitopes / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011991:Receptors, Virus / Q000235:genetics / Q000502:physiology*; D015183:Restriction Mapping; D012229:Rhinovirus / Q000502:physiology*; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D011991:Receptors, Virus; D018799:Intercellular Adhesion Molecule-1