A protein binding to a minor-group human rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino acid sequences of tryptic peptides showed identity with the human low density lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding sites on human fibroblasts. Cells down-regulated for LDLR expression yielded much less HRV2 upon infection than cells with up-regulated LDLR. Virus also bound to the large subunit of the a2-macroglobulin receptor/LDLRrelated protein (a2MR/LRP). LDLR-deficient fibroblasts yielded considerably less virus in the presence of receptorassociated protein (RAP), providing evidence that a2MR/LRP also acts as a minor group HRV receptor.Common colds most frequently arise through infection with human rhinoviruses (HRVs). The 102 antigenically distinct serotypes are divided into two groups based on receptor specificity (1, 2). The major group binds to the intercellular adhesion molecule 1 (ICAM-1) (3)(4)(5), and the minor group has been shown to attach to a membrane protein with a relative molecular mass of about 120 kDa (6, 7). ICAM-1 and the poliovirus receptor (8) are members of the immunoglobulin superfamily. As the three-dimensional structures of representative HRVs from the two different receptor groups (9, 10) and of poliovirus (11) show considerable similarity, it might have been expected that the minor group receptor would also belong to this family. However, in this communication we present evidence that minor-group HRVs gain access to the cell via members of the low density lipoprotein (LDL) receptor (LDLR) family (12,13). MATERIALS AND METHODSPurification of HRV2-Binding Protein. Two hundred liters of HeLa cell culture supernatant were concentrated ten times by ultrafiltration, dialyzed against 250 liters of H20 containing 0.02% NaN3, and adjusted to contain 20 mM N-methylpiperazine hydrochloride (pH 4.5). Precipitated material was removed, and the filtered supernatant was applied to a 0.5-liter Macroprep 50 Q column (Bio-Rad). Bound material was eluted with the same buffer containing 0.5 M NaCl. After adjustment to pH 7.2 with 1 M Tris HCl (pH 8), the material was loaded onto a 100-ml Lens culinaris lectin column (Pharmacia), and bound protein was eluted with phosphatebuffered saline (PBS) containing 0.5 M a-D-methyl glucopyranoside and precipitated with (NH4)2SO4 at 50o saturation. The precipitate was dissolved in 200 ml of PBS, the solution was passed over a 40-ml Jacalin agarose column (Vector Laboratories), and bound protein was eluted with 120 ml of 0.1 M a-D-methyl galactopyranoside in PBS and precipitated with (NH4)2SO4 as above. The precipitate was dissolved in 20 mM N-methylpiperazine hydrochloride (pH 4.5) and desalted on a PD-10 column (Pharmacia). Protein was applied onto a Mono Q HR 5/5 column (Pharmacia) and eluted with a gradient of 0-0.5 M NaCl in the same buffer. The binding activity was monitored throughout the purification procedure on ligand blots (7). Active fractions were concentrated to 1.5 ml with a Centricon-30...

A fragment of the low density lipoprotein receptor encompassing the seven ligand binding repeats was expressed in Sf9 insect cells as a fusion protein with a carboxyl-terminally linked hexa-his tag by using a baculovirus vector. Up to 10 mg/l of the fusion protein was secreted into the medium. The material was soluble in the absence of detergent and active in binding beta very low density lipoprotein and a member of the minor group of human rhinoviruses (HRV2) in ligand blots from sodium dodecyl sulfate-polyacrylamide gels run under nonreducing conditions. The receptor fragment specifically inhibits viral infection of HeLa cells by minor group HRVs in a concentration-dependent manner. Viral infectivity is neutralized by aggregation.

Four WIN compounds with anti-picornavirus activities were tested for their ability to stabilize human rhinovirus serotype 2 (HRV-2) against low pH-induced conformational changes in vitro, as determined by specific immunoprecipitation. These results were compared to the minimal inhibitory concentration (MIC) as measured in a plaque reduction assay. A direct relationship was observed between the concentration of the compound that prevented the low pH-induced conformational changes and the MIC, indicating that stabilization is an important element in the mode of action of these drugs against HRV-2.

Avian oocyte-specific very low density lipoprotein receptor specifically binds human rhinovirus of the minor receptor group on ligand blots and in solution. The solubilized receptor protects cells against infection in a dose-dependent manner.

No abstract