Viral Evolution toward Change in Receptor Usage: Adaptation of a Major Group Human Rhinovirus To Grow in ICAM-1-Negative Cells

Abstract

Major receptor group common cold virus HRV89 was adapted to grow in HEp-2 cells, which are permissive for minor group human rhinoviruses (HRVs) but which only marginally support growth of major-group viruses. After 32 blind passages in these cells, each alternating with boosts of the recovered virus in HeLa cells, HRV89 acquired the capacity to effectively replicate in HEp-2 cells, attaining virus titers comparable to those in HeLa cells although no cytopathic effect was observed. Several clones were isolated and shown to replicate in HeLa cells whose ICAM-1 was blocked with monoclonal antibody R6.5 and in COS-7 cells, which are devoid of ICAM-1. Blocking experiments with recombinant very-low-density lipoprotein receptor fragments and enzyme-linked immunosorbent assays indicated that the mutants bound a receptor different from that used by minor-group viruses. Determination of the genomic RNA sequence encoding the capsid protein region revealed no changes in amino acid residues at positions equivalent to those involved in the interaction of HRV14 or HRV16 with ICAM-1. One mutation was within the footprint of a very-low-density lipoprotein receptor fragment bound to minor-group virus HRV2. Since ICAM-1 not only functions as a vehicle for cell entry but has also a "catalytic" function in uncoating, the use of other receptors must have important consequences for the entry pathway and demonstrates the plasticity of these viruses.Human rhinoviruses (HRVs), a major cause of mild upper respiratory infections generally recognized as common colds, are small icosahedral particles with a capsid composed of four viral proteins, VP1 through VP4 (for a review see reference 9). The capsid encases a genomic RNA of about 7,500 nucleotides encoding a polyprotein which is cotranslationally and autocatalytically processed by three viral proteinases, P2A, P3C, and P3CD (the precursor of P3C). A final maturation cleavage of VP0 to VP2 and VP4 occurs concomitantly with encapsidation by an as yet unidentified protease. With one exception (HRV87), the serotypes can be divided into a major group, using intercellular adhesion molecule 1 (ICAM-1) as the viral receptor, and a minor group, attaching to the cell via members of the low-density lipoprotein receptor (LDLR) family including LDLR, the very-low-density lipoprotein receptor (VLDLR), and LDLR-related protein (LRP) (16,27). The nature of the HRV87 receptor is unknown (50). Whereas major-group viruses are highly specific for human ICAM-1 and fail to attach to the homologue of other species, minor-group viruses bind to a variety of LDLRs, most likely due to the high evolutionary conservation of these membrane proteins. Replication usually does not occur in nonhuman cells even when suitable receptors are present, and adaptation of HRV2 to growth in mouse cells has been shown to be correlated with mutations in nonstructural proteins P2B and P2C (23).As HRVs of both receptor groups are very similar with respect to the amino acid sequence and the three-dimensional structure of the viral cap...

Species-Specific Receptor Recognition by a Minor-Group Human Rhinovirus (HRV): HRV Serotype 1A Distinguishes between the Murine and the Human Low-Density Lipoprotein Receptor

“…Comparison of the footprint of the VLDLR on HRV2 with that predicted for HRV1A, based on the amino acid sequence and the X-ray structure of this serotype, revealed only marginal similarity. As noted earlier (8,21,40), no more than the tripeptide TEK in the HI loop and the dipeptide YN in the BC loop are conserved within all minorgroup serotypes sequenced so far. However, in contrast to foot-and-mouth disease virus, where the interaction between the RGD tripeptide in the GH loop of VP1 with ␣ v ␤ 3 integrin (2) can be inhibited with synthetic peptides (10), the interaction of HRVs with their receptors cannot be blocked by a peptide including the TEK sequence (our unpublished observations).…”

The Structure of Echovirus Type 12 Bound to a Two-domain Fragment of Its Cellular Attachment Protein Decay-accelerating Factor (CD 55)

“…Therefore, we are forced to consider the possibility that the different modes of binding in these viruses may represent a comparatively recent adaptation of a preexisting trait on the part of one or other virus. Rapid evolution of novel receptor binding mechanisms is found throughout the picornaviruses, such as culture adaptation to bind heparan sulfate in foot and mouth disease virus, the in vitro evolution of rhinoviruses capable of infecting cells in an ICAM-1-independent manner, and the reversion of non-DAF binding EV11 mutants to a DAF binding phenotype (29,52,53). Although the evolution of a novel receptor binding site might seem intuitively improbable for a virus that has an already functional interaction with this receptor, consideration of the high mutation and recombination rates in these viruses as well as the numbers of virions produced in the course of an infection makes such events plausible.…”

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

“…Parts of the alignment of the entire VP1 sequences made with Clustal W (http://www.ebi.ac.uk/clustalw/index.html#) are shown. this type of assay, most probably because of the presence of SDS (24). No signal was seen when the blot was incubated with the HRVs in the absence of Ca 2ϩ but in the presence of 10 mM EDTA, indicating the specificity of the assay (data not shown).…”

“…e Hep2-cell adapted mutant HRV89 15 using proteoglycan receptors (24,38). Table 2, the four minor group HRVs (including HRV23 and HRV25) tested were inhibited by MBP-V33333 but not by R6.5.…”

“…Transfection with an ICAM-1-encoding plasmid restored virus binding (29). We have previously used COS-7 cells to demonstrate that a variant of HRV89 adapted to grow in HEp-2 cells (which express only very low levels of ICAM-1) had acquired a new receptor specificity and replicated in COS-7 cells but at the same time retained its affinity for ICAM-1 (24). COS-7 cells were lysed upon infection with HRV2, HRV23, and HRV25 (Fig.…”