Biochemical characterization of a glycoprotein required for rhinovirus attachment

Human rhinoviruses attach to specific receptors located on the surfaces of host cells as a first step in viral infection. A 90-kDa cell surface protein was previously shown to be involved in the attachment of human rhinoviruses to susceptible cells (Tomassini, J. E., and Colonno, R.J. (1986) J. Virol. 58, 290-295). Digestion of purified receptor protein with various glycosidases revealed that 30% of its molecular mass was comprised of complex-type oligosaccharides, one-third being contributed by sialic acid. The presence of sialic acid was confirmed by demonstrating that wheat germ lectin can inhibit the attachment of rhinoviruses to host cell membranes, while lectins of other sugar specificities had no effect. The oligosaccharides were shown to be N-linked by tunicamycin treatment of host cells and by N-glycanase digestion. Seven N-linked glycosylation sites were detected by partial digestion of the receptor oligosaccharides with N-glycanase. Native receptor protein had an isoelectric focusing point of 4.2, compared to 5.3 for the deglycosylated protein. Studies of virus and antibody binding to neuraminidase-treated host cell membranes suggested that although carbohydrates may be involved in host-virus interaction, the receptor carbohydrate is not the predominant component of the cellular receptor site.     

Two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity.

A variety of chemically different compounds inhibit the replication of several serotypes of rhinoviruses (common-cold viruses). We noticed that one of these antiviral compounds, WIN 51711, had an antiviral spectrum clearly distinctive from a consensus spectrum or other capsid-binding compounds, although all of them were shown to share the same binding site. A systematic evaluation of all known rhinovirus capsid-binding compounds against all serotyped rhinoviruses was therefore initiated. Multivariate analysis of the results revealed the existence of two groups of rhinoviruses, which we will call antiviral groups A and B. The differential sensitivity of members of these groups to antiviral compounds suggests the existence of a dimorphic binding site. The antiviral groups turned out to be a reflection of a divergence of rhinovirus serotypes on a much broader level. Similarities in antiviral spectra were highly correlated with sequence similarities, not only of amino acids lining the antiviral compound-binding-site, but also of amino acids of the whole VP1 protein. Furthermore, analysis of epidemiological data indicated that group B rhinoviruses produced more than twice as many clinical infections per serotype than group A rhinoviruses did. Rhinoviruses belonging to the minor receptor group were without exception all computed to lie in the same region of antiviral group B.     

A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses

Rhinoviruses, which cause common colds, possess over 100 serotypes, 90% of which (the major group) share a single receptor. Lymphocyte function associated molecule 1 (LFA-1) mediates leukocyte adhesion to a wide variety of cell types by binding to intercellular adhesion molecule 1 (ICAM-1). We demonstrate identity between the receptor for the major group of rhinoviruses and ICAM-1. A major group rhinovirus binds specifically to purified ICAM-1 and to ICAM-1 expressed on transfected COS cells, and binding is blocked by three ICAM-1 monoclonal antibodies (MAb) that block ICAM-1-LFA-1 interaction, but not by an ICAM-1 MAb that does not block ICAM-1-LFA-1 interaction. This suggests that the ICAM-1 contact site(s) for LFA-1 and rhinoviruses is proximal or identical. In addition, ICAM-1 MAb block the cytopathic effect in HeLa cells mediated by representative major but not minor group rhinoviruses. ICAM-1 is induced by soluble mediators of inflammation, suggesting that the host immune response to rhinovirus may facilitate spread to uninfected cells.     

Distinct cellular receptor interactions in poliovirus and rhinoviruses

Receptor binding to human poliovirus type 1 (PV1/M) and the major group of human rhinoviruses (HRV) was studied comparatively to uncover the evolution of receptor recognition in picornaviruses. Surface plas- mon resonance showed receptor binding to PV1/M with faster association and dissociation rates than to HRV3 and HRV16, two serotypes that have similar binding kinetics. The faster rate for receptor association to PV1/M suggested a relatively more accessible binding site. Thermodynamics for receptor binding to the viruses and assays for receptor-mediated virus uncoating showed a more disruptive receptor interaction with PV1/M than with HRV3 or HRV16. Cryo-electron microscopy and image reconstruction of receptor-PV1/M complexes revealed receptor binding to the 'wall' of surface protrusions surrounding the 'canyon', a depressive surface in the capsid where the rhinovirus receptor binds. These data reveal more exposed receptor-binding sites in poliovirus than rhinoviruses, which are less protected from immune surveillance but more suited for receptor-mediated virus uncoating and entry at the cell surface.     

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo.     

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.     

CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7

The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7.     

Review: rhinoviruses and their ICAM receptors

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen or macrophage-1 antigen. However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses.     

Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1

Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.     

Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses.

Reciprocal competition binding assays have previously demonstrated that 20 of 24 human rhinovirus serotypes tested compete for a single cellular receptor. These studies suggested that the vast majority of rhinovirus serotypes utilize a single cellular receptor. With HeLa cells as an immunogen, a mouse monoclonal antibody was isolated which had the precise specificity predicted by the competition binding study. The receptor antibody was shown to protect HeLa cells from infection by 78 of 88 human rhinovirus serotypes assayed. In addition, the receptor antibody protects HeLa cells from infection by three coxsackievirus A serotypes. The receptor antibody was unable to protect cells from infection by a wide range of other RNA and DNA viruses. Using the receptor antibody and human rhinovirus type 15, we determined that the cellular receptor utilized by the vast number of human rhinovirus serotypes is present only on cells of human origin, with the exception of chimpanzee-derived cells. The receptor antibody has a strong affinity for the cellular receptor as evidenced by its rapid binding kinetics and ability to displace previously bound human rhinovirus virions from receptors. No viral variants were identified which could bypass the receptor blockage.     

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

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore "true" major group viruses.     

Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection

The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341-1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.     

Sequence and structure of human rhinoviruses reveal the basis of receptor discrimination

The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.     

The crystal structure of coxsackievirus A21 and its interaction with ICAM-1

CVA21 and polioviruses both belong to the Enterovirus genus in the family of Picornaviridae, whereas rhinoviruses form a distinct picornavirus genus. Nevertheless, CVA21 and the major group of human rhinoviruses recognize intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, whereas polioviruses use poliovirus receptor. The crystal structure of CVA21 has been determined to 3.2 A resolution. Its structure has greater similarity to poliovirus structures than to other known picornavirus structures. Cryo-electron microscopy (cryo-EM) was used to determine an 8.0 A resolution structure of CVA21 complexed with an ICAM-1 variant, ICAM-1(Kilifi). The cryo-EM map was fitted with the crystal structures of ICAM-1 and CVA21. Significant differences in the structure of CVA21 with respect to the poliovirus structures account for the inability of ICAM-1 to bind polioviruses. The interface between CVA21 and ICAM-1 has shape and electrostatic complementarity with many residues being conserved among those CVAs that bind ICAM-1.     

Isolation of a receptor protein involved in attachment of human rhinoviruses

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.     

VP1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds

Rhinoviruses are the most common infectious agents of humans. They are the principal etiologic agents of afebrile viral upper-respiratory-tract infections (the common cold). Human rhinoviruses (HRVs) comprise a genus within the family Picornaviridae. There are >100 serotypically distinct members of this genus. In order to better understand their phylogenetic relationship, the nucleotide sequence for the major surface protein of the virus capsid, VP1, was determined for all known HRV serotypes and one untyped isolate (HRV-Hanks). Phylogenetic analysis of deduced amino acid sequence data support previous studies subdividing the genus into two species containing all but one HRV serotype (HRV-87). Seventy-five HRV serotypes and HRV-Hanks belong to species HRV-A, and twenty-five HRV serotypes belong to species HRV-B. Located within VP1 is a hydrophobic pocket into which small-molecule antiviral compounds such as pleconaril bind and inhibit functions associated with the virus capsid. Analyses of the amino acids that constitute this pocket indicate that the sequence correlates strongly with virus susceptibility to pleconaril inhibition. Further, amino acid changes observed in reduced susceptibility variant viruses recovered from patients enrolled in clinical trials with pleconaril were distinct from those that confer natural phenotypic resistance to the drug. These observations suggest that it is possible to differentiate rhinoviruses naturally resistant to capsid function inhibitors from those that emerge from susceptible virus populations as a result of antiviral drug selection pressure based on sequence analysis of the drug-binding pocket.     

Very-Low-Density Lipoprotein Receptor Fragment Shed from HeLa Cells Inhibits Human Rhinovirus Infection

The large family of human rhinoviruses, the main causative agents of the common cold, is divided into the major and the minor group based on receptor specificity. Major group viruses attach to intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily, whereas minor group viruses use low-density lipoprotein receptors (LDLR) for cell entry. During early attempts aimed at isolating the minor group receptor, we discovered that a protein with virus binding activity was released from HeLa cells upon incubation with buffer at 37°C (F. Hofer, B. Berger, M. Gruenberger, H. Machat, R. Dernick, U. Tessmer, E. Kuechler, and D. Blaas, J. Gen. Virol. 73:627–632, 1992). In light of the recent discovery of several new members of the LDLR family, we reinvestigated the nature of this protein and present evidence for its being derived from the human very-low density lipoprotein receptor (VLDLR). A soluble VLDLR fragment encompassing the eight complement type repeats and representing the N-terminal part of the receptor was then expressed in the baculovirus system; both the shed protein and the recombinant soluble VLDLR bind minor group viruses and inhibit viral infection of HeLa cells in a concentration-dependent manner.     

The Coxsackievirus-Adenovirus Receptor Protein Can Function as a Cellular Attachment Protein for Adenovirus Serotypes from Subgroups A, C, D, E, and F

Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein. To date, only the cellular fiber receptor for subgroup C serotypes 2 and 5, the so-called coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. Previous data suggested that the fiber of the subgroup D serotype Ad9 also recognizes CAR, since Ad9 and Ad2 fiber knobs cross-blocked each other’s cellular binding. Recombinant fiber knobs and ³H-labeled Ad virions from serotypes representing all six subgroups (A to F) were used to determine whether the knobs cross-blocked the binding of virions from different subgroups. With the exception of subgroup B, all subgroup representatives cross-competed, suggesting that they use CAR as a cellular fiber receptor as well. This result was confirmed by showing that CAR, produced in a soluble recombinant form (sCAR), bound to nitrocellulose-immobilized virions from the different subgroups except subgroup B. Similar results were found for blotted fiber knob proteins. The subgroup F virus Ad41 has both short and long fibers, but only the long fiber bound sCAR. The sCAR protein blocked the attachment of all virus serotypes that bound CAR. Moreover, CHO cells expressing human CAR, in contrast to untransformed CHO cells, all specifically bound the sCAR-binding serotypes. We conclude therefore that Ad serotypes from subgroups A, C, D, E, and F all use CAR as a cellular fiber receptor.