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Minor group human rhinoviruses (HRVs) bind members of the low-density lipoprotein receptor family for cell entry. The ligand-binding domains of these membrane proteins are composed of various numbers of direct repeats of about 40 amino acids in length. Residues involved in binding of module 3 (V3) of the very-low-density lipoprotein receptor (VLDLR) to HRV2 have been identified by X-ray crystallography (N. Verdaguer, I. Fita, M. Reithmayer, R. Moser, and D. Blaas, Nat. Struct. Mol. Biol. 11: 429-434, 2004). Sequence comparisons of the eight repeats of VLDLR with respect to the residues implicated in the interaction between V3 and HRV2 suggested that (in addition to V3) V1, V2, V5, and V6 also fulfill the requirements for interacting with the virus. Using a highly sensitive binding assay employing phage display, we demonstrate that single modules V2, V3, and V5 indeed bind HRV2. However, V1 does not. A single mutation from threonine 17 to proline converted the nonbinding wild-type form of V1 into a very strong binder. We interpret the dramatic increase in affinity by the generation of a hydrophobic patch between virus and receptor; in the presence of threonine, the contact area might be disturbed. This demonstrates that the interaction between virus and its natural receptors can be strongly enhanced by mutation.

Human rhinoviruses (HRVs), the main causative agents of common cold, were originally classified as acid-sensitive picornaviruses (34). 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). HRV87 was noted to be an exception in that it binds a sialylated membrane protein. Based on comparison of nucleotide sequences in several genomic regions, HRV87 was subsequently classified as an acid-sensitive enterovirus and found to be a prime strain of enterovirus 68 that uses decay-accelerating factor as a receptor (2, 27). By phylogenetic analysis of capsid protein VP4/VP2 coding sequences of all HRV prototype strains, 74 HRV serotypes, including all the minor receptor group ones, were classified as HRV species A, while the 25 remaining serotypes form HRV species B (27). In a paper from the Colonno group, primary data on the competition with an ICAM-1-blocking antibody were not explicitly presented for all serotypes, in particular not for HRV23 and HRV25, and the presumed allocation of all HRVs to either group was depicted only in the form of a summarizing figure (35). Despite this, it was generally accepted that 90 serotypes, including HRV23 and HRV25, were major group viruses. Later, Crump and colleagues reported that recombinant soluble ICAM-1 and a chimeric ICAM-1/immunoglobulin A molecule failed to neutralize these two serotypes and concluded that they most probably use a receptor different from the major and minor group receptors (4, 5). This finding remained largely unnoticed by the picornavirus community. As soon as the capsid protein VP1 sequences of all human rhinoviruses were determined (15, 16), we became aware of genetic clustering of HRV23 and HRV25 close to certain minor receptor group serotypes and of the presence of a lysine in the HI loop. This residue is strictly conserved in all minor group viruses and is believed to be essential but not sufficient for attachment to members of the LDLR family (37). A lysine is also present at the equivalent position in HRV8, -18, -24, -40, -54, -56, -58, -85, -95, and -98. However, these latter serotypes had been explicitly shown to be neutralized by soluble recombinant ICAM-1 (5). Therefore, we wondered whether at least HRV23 and HRV25 might use members of the LDLR family as receptors. We also wondered whether the other HRVs containing the conserved lysine might eventually possess dual receptor specificity and attach to ICAM-1 as well as to LDLR. The ligand binding domain at the N terminus of LDLR that is implicated in binding minor group HRVs is composed of seven imperfect direct repeats of roughly 40 amino acid residues in length. Three disulfide bridges and a Ca2+ ion within an octagonal cage formed by the carboxylates of three aspartates and one glutamate, together with two backbone oxygens, stabilize this structure (7). The very-low-density lipoprotein receptor (VLDLR) and LDLR-related protein (LRP) possess 8 and 31 such modules, respectively (30). In LRP the modules are not contiguous but are arranged in clusters of 2, 8, 10, and 11. We have previously shown that a soluble recombinant fusion protein composed of the maltose binding protein (MBP) and an artificial concatemer of five copies of repeat 3 (V3) of VLDLR arranged in tandem (MBP-V33333) exhibits strong virus neutralization capacity toward the 10 minor group HRVs (36). Therefore, by using cell protection assays with this protein, ligand blots, and replication in COS-7 cells that do not express ICAM-1, we investigated the nature of the strains containing a lysine residue in the HI loop and found that HRV23 and HRV25 possess all characteristics of minor group viruses. Examination of the major group serotypes with the lysine in the HI loop as mentioned above showed that none was able to use both ICAM-1 and LDLR for cell binding. Therefore, out of the numbered 99 HRV serotypes, altogether, 12 serotypes (if we count HRV1A and -B separately) belong to the minor receptor group.

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76: 6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDG GP D in the acidic cluster of module 5 in the human receptor to CDG EA D present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.

Human rhinoviruses (HRV) of the minor receptor group use several members of the low-density lipoprotein receptor superfamily for cell entry. These proteins are evolutionarily highly conserved throughout species and are almost ubiquitously expressed. Their common building blocks, cysteine-rich ligand binding repeats about 40 amino acids in length, exhibit considerable sequence similarity. Various numbers of these repeats are present in the different receptors. We here demonstrate that HRV type 1A (HRV1A) replicates in mouse cells without adaptation. Furthermore, although closely related to HRV2, it fails to bind to the human low-density lipoprotein receptor but recognizes the murine protein, whereas HRV2 binds equally well to both homologues. This difference went unnoticed due to the presence of other receptors, such as the low-density lipoprotein receptor-related protein, which allow species-independent attachment. The species specificity of HRV1A reported here will aid in defining amino acid residues establishing the contact between the viral surface and the receptor.

HeLa cells were stably transfected with a cDNA clone encoding the B1 isoform of the mouse FcγRII receptor (hereafter referred to as HeLa-FcRII cells). The receptor was expressed at high level at the plasma membrane in about 90% of the cells. These cells bound and internalized mouse monoclonal virus-neutralizing antibodies 8F5 and 3B10 of the subtype immunoglobulin G2a (IgG2a) and IgG1, respectively. Binding of the minor-group human rhinovirus type 2 (HRV2) to its natural receptors, members of the low-density lipoprotein receptor family, is dependent on the presence of Ca 2+ ions. Thus, chelating Ca 2+ ions with EDTA prevented HRV2 binding, entry, and infection. However, upon complex formation of 35 S-labeled HRV2 with 8F5 or 3B10, virus was bound, internalized, and degraded in HeLa-FcRII cells. Furthermore, challenge of these cells with HRV2-8F5 or HRV2-3B10 complexes resulted in de novo synthesis of viral proteins, as shown by indirect immunofluorescence microscopy. These data demonstrate that minor-group receptors can be replaced by surrogate receptors to mediate HRV2 cell entry, delivery into endosomal compartments, and productive uncoating. Consequently, the conformational change and uncoating of HRV2 appears to be solely triggered by the low-pH (pH ≤ 5.6) environment in these compartments.

The very-low-density lipoprotein receptor (VLDL-R) is a receptor for the minor-group human rhinoviruses (HRVs). Only two of the eight binding repeats of the VLDL-R bind to HRV2, and their footprints describe an annulus on the dome at each fivefold axis. By studying the complex formed between a selection of soluble fragments of the VLDL-R and HRV2, we demonstrate that it is the second and third repeats that bind. We also show that artificial concatemers of the same repeat can bind to HRV2 with the same footprint as that for the native receptor. In a 16-Å-resolution cryoelectron microscopy map of HRV2 in complex with the VLDL-R, the individual repeats are defined. The third repeat is strongly bound to charged and polar residues of the HI and BC loops of viral protein 1 (VP1), while the second repeat is more weakly bound to the neighboring VP1. The footprint of the strongly bound third repeat extends down the north side of the canyon. Since the receptor molecule can bind to two adjacent copies of VP1, we suggest that the bound receptor “staples” the VP1s together and must be detached before release of the RNA can occur. When the receptor is bound to neighboring sites on HRV2, steric hindrance prevents binding of the second repeat.