CDHR3 extracellular domains EC1-3 mediate rhinovirus C interaction with cells and as recombinant derivatives, are inhibitory to virus infection

Abstract

Viruses in the rhinovirus C species (RV-C) are more likely to cause severe wheezing illnesses and asthma exacerbations in children than related isolates of the RV-A or RV-B. The RV-C capsid is structurally distinct from other rhinoviruses and does not bind ICAM-1 or LDL receptors. The RV-C receptor is instead, human cadherin-related family member 3 (CDHR3), a protein unique to the airway epithelium. A single nucleotide polymorphism (rs6967330, encoding C529Y) in CDHR3 regulates the display density of CDHR3 on cell surfaces and is among the strongest known genetic correlates for childhood virus-induced asthma susceptibility. CDHR3 immunoprecipitations from transfected or transduced cell lysates were used to characterize the RV-C interaction requirements. The C529 and Y529 variations in extracellular repeat domain 5 (EC5), bound equivalently to virus. Glycosylase treatment followed by mass spectrometry mapped 3 extracellular N-linked modification sites, and further detected surface-dependent, α2–6 sialyation unique to the Y529 format. None of these modifications were required for RV-C recognition, but removal or even dilution of structurally stabilizing calcium ions from the EC junctions irreversibly abrogated virus binding. CDHR3 deletions expressed in HeLa cells or as bacterial recombinant proteins, mapped the amino-terminal EC1 unit as the required virus contact. Derivatives containing the EC1 domain, could not only recapitulate virus:receptor interactions in vitro, but also directly inhibit RV-C infection of susceptible cells for several virus genotypes (C02, C15, C41, and C45). We propose that all RV-C use the same EC1 landing pad, interacting with putative EC3-mediated multimerization formats of CDHR3.

Cadherin-related family member 3 (CDHR3) drives differentiation of ciliated bronchial epithelial cells and facilitates rhinovirus C infection, although with a little help

“…Contrary to earlier reports, [3][4][5][7][8][9] in which CDHR3 mediated both rhinovirus C binding and infection of cells, Everman et al 6 found that CDHR3 knockdown did not affect binding of rhinovirus C, whereas it affected rhinovirus C replication. The underlying reason for these apparent discordant results is not clear.…”

“…Modeling of the CDHR3 structure suggested potential binding sites for the capsid proteins VP1 and VP2 of rhinovirus C. Mapping of the rhinovirus C binding site or sites on CDHR3 revealed that extracellular domain 1 is the likely docking site for all rhinovirus C types, which was also supported by the capacity of extracellular domain 1-containing proteins/peptides to inhibit rhinovirus C infection of susceptible cells. 4 Sequence analysis of a rhinovirus C strain that displayed improved binding and replication in in vitro cultures showed that a mutation in the viral capsid protein VP1 was indeed responsible for the improved binding. 5 Together, these data are highly suggestive of CDHR3 being the docking site for rhinovirus C types, but independent confirmation is still required.…”

Cryo-EM structure of rhinovirus C15a bound to its cadherin-related protein 3 receptor

“…The archetype adhesion function of classic cadherins is achieved by homotypic or heterotypic EC1 strand-swapping requiring one or two N-proximal tryptophan residue(s) (33,34). EC1 and EC1+2 recombinant proteins are monomers in solution (24), and there is no homologous N-proximal tryptophan, so CDHR3 must necessarily use an alternative strategy for adhesive binding (35). The new structure shows that the upper face of EC1 away from the virus has a deep groove-like feature between strands D and C ( Fig.…”

“…Each repeat sequence is consistent with a cadherin seven-strand β-sandwich fold. The native protein has three mapped N-linked glycosylation sites (N186, N384, and N624) which become further sialylated when the Y529 variant is expressed in tissue culture (24). This particular residue, at the junction of the EC5 and EC6 repeats, presumably helps regulate structurally stabilizing interdomain Ca 2+ binding, without which cadherins are improperly trafficked and/or withdrawn from cell surfaces (25)(26)(27).…”

“…The EC2 and EC3 repeats influence this only insofar as some engineered mutations there have the potential to disrupt native protein folding. Soluble recombinant EC1, properly refolded in the presence of Ca 2+ , recapitulates virus-receptor interactions in vitro and can directly inhibit RV-C infection of susceptible cells for several viral genotypes (24).…”