Abstract
Soluble human low density lipoprotein minireceptors with less than seven ligand binding repeats (as are present in the native membrane receptor) were expressed in Sf9 insect cells with a hexa-His tag fused to the C terminus. The recombinant truncated proteins were affinity purified from the tissue culture supernatants by Ni-NTA column chromatography. Minireceptors with more than two repeats bound to rabbit beta very low density lipoprotein and could thus be further purified by affinity chromatography. Binding and cell protection assays indicated that two ligand binding repeats are sufficient for attachment of minor group human rhinoviruses to immobilized receptors, whereas at least three ligand binding repeats are required to protect HeLa cells against viral infection.
Highlights
With 102 serotypes, human rhinoviruses (HRVs)1 constitute the largest genus within the family Picornaviridae
We have shown that a recombinant fragment of human very low density lipoprotein receptor, which contains eight complement type repeats, binds minor group HRVs as well [9]
We have previously shown that human rhinovirus serotype 2 (HRV2) attaches to low density lipoprotein receptor (LDLR), LDLR-related protein (LRP) [7], vitellogenin receptor [8], recombinant soluble human very low density lipoprotein receptor (VLDLR) [9], and recombinant soluble rLDLR1-7h [10] in ligand blots from SDS-polyacrylamide gels run under nonreducing conditions
Summary
With 102 serotypes, human rhinoviruses (HRVs) constitute the largest genus within the family Picornaviridae. The most prominent common structural motive of all members of the LDLR family are various numbers of complement type-A repeats, about 40 amino acids in length, containing six cysteines each, which are involved in disulfide bridges; the C termini exhibit clusters of negatively charged amino acid residues. These repeats form the ligand binding domain and interact with a great number of structurally and functionally unre-. We show that fragments with two or more complement type repeats are recognized by minor group HRVs when immobilized on PVDF membranes and that the presence of more than two repeats is required to inhibit viral infection of HeLa cells
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