Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication

Abstract

Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared with other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3) enables the cells normally unsusceptible to RV-C infection to support both virus binding and replication. A coding single nucleotide polymorphism (rs6967330, C529Y) was previously linked to greater cell-surface expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared with wild-type CDHR3, cells transfected with the CDHR3-Y529 variant had about 10-fold increases in RV-C binding and progeny yields. We developed a transduced HeLa cell line (HeLa-E8) stably expressing CDHR3-Y529 that supports RV-C propagation in vitro. Modeling of CDHR3 structure identified potential binding sites that could impact the virus surface in regions that are highly conserved among all RV-C types. Our findings identify that the asthma susceptibility gene product CDHR3 mediates RV-C entry into host cells, and suggest that rs6967330 mutation could be a risk factor for RV-C wheezing illnesses.

Keywords: CDHR3; receptor; rhinovirus C.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Identification of candidate RV-C receptors by gene expression analysis. (A) Venn diagrams showing the common receptor candidate genes identified in two series of microarray experiments after the filtering steps. The differentially expressed genes were filtered stepwise on the basis of the indicated criteria and the diagrams were generated by ArrayStar software v4.0 (DNASTAR). (B) Heat map showing clustering analysis of expression profiles of the 12 differentially expressed genes in primary cells, cell lines, and whole-tissue samples. Candidate genes selected for validation of RV-C receptor activity are shown in red. Expression intensity values were analyzed by hierarchical clustering of samples and genes using the Euclidean distance metric with centroid linkage method using ArrayStar software v4.0 (DNASTAR). Color bar represents gene expression intensity (log2 scale). See also Figs. S1 and S2 and Table S1.

Fig. 2.

RV-C15 replication in HeLa cells transfected with CDHR3 cDNA. (A) Genome structure of the RV-C15 infectious clone expressing GFP. Two viral 2A protease cleavage sites permitting the release of GFP after polyprotein translation are indicated. (B) GFP expression 48 h after C15-GFP infection of HeLa cells transfected with wild-type CDHR3 for 48 h. (Scale bar, 100 µm.) Representative image of three independent experiments. (C) RV-C15 binding (2 hpi) and replication (48 hpi) in HeLa cells transfected with wild-type CDHR3 (ο), or Lipofectamine 2000 control (∆) for 48 h (n = 5, data are means ± SD). (D) Fluorescent microscopy images of cells transfected with FLAG-tagged versions of CDHR3 wild-type (C529) or Cys529→Tyr variant (Y529). Permeabilized (cytoplasm) or nonpermeabilized (surface) HeLa cells were stained with the α-CDHR3 or α-FLAG antibodies, respectively, to visualize intracellular or surface expression. (Scale bar, 100 µm.) (E) Western blot analysis of CDHR3 expression in HeLa cells transfected with wild-type (C529) or Cys529→Tyr variant (Y529) for 24 h and probed with α-CDHR3 polyclonal antibodies and α-tubulin antibodies (loading control). (F) GFP expression 48 h after C15-GFP infection of HeLa cells transfected with CDHR3 Cys529→Tyr variant for 48 h. (Scale bar, 100 µm.) Representative image of three independent experiments. (G) RV-C15 binding (2 hpi) and replication (48 hpi) in HeLa cells transfected with CDHR3 Cys529→Tyr variant (□), FLAG-tagged CDHR3 Cys529→Tyr (ο), or Lipofectamine 2000 control (∆) for 48 h (n = 5, data are means ± SD). See also Figs. S3 and S4.

Fig. 3.

RV-C binding and replication in HeLa-E8 cells stably expressing CDHR3-Y529. (A) Binding (2 hpi) and replication (24 hpi) of RV-C clinical isolates in HeLa-E8 cells. (B) CDHR3 mRNA and protein expression in transduced (HeLa-E8) compared with control HeLa cells assessed by RT-qPCR and Western blot analysis. For mRNA expression, each bar represents five different RNA preparations (n = 5) and data are presented as fold-change relative to control cells. For protein analysis, whole-cell lysates were probed with α-CDHR3 polyclonal antibodies. (C) Control and transduced (HeLa-E8) cells were fixed, permeabilized, stained with monoclonal anti-CDHR3 antibody (Abcam, ab56549), and analyzed by flow cytometry. Data for control and transduced cells were acquired separately and combined. Data are representative of three independent experiments. (D) Growth curve of RV-C15, RV-A16 and RV-B52 clinical isolates in HeLa-E8 cells (n = 3, data are means ± SD). (E) Binding characteristics of fluorescently labeled RV-C15-APC in control and transduced (HeLa-E8) cells analyzed by flow cytometry. The percentages shown in the top two quadrants represent the percent of gated events that are negative for GFP fluorescence but positive for APC fluorescence (Left) or positive for both GFP and APC fluorescence (Right). Data are representative of three independent experiments.

Fig. 4.

Structure modeling of RV-C15 binding to CDHR3 receptor. (A) Schematic map of the CDHR3 protein with indicated domains, predicted glycosylation sites, and engineered point mutation and FLAG tag. (B) Structural model of the complete CDHR3 protein. Domain 1 was added to the published model of domains 2–6 (19) after I-Tasser modeling relative to known cadherin structures. Key residues are highlighted. Gray spheres are interdomain calcium ions from PDB ID code 3Q2W. (C) Surface model of domains 1–3 docked to two RV-C15 protomers (model), projected onto pentamer coordinates. VP1 (blue), VP2 (green), and VP3 (red) are shown following standard colors. Model was then duplicated across twofold axis to show putative paired orientations. Triangle marks the icosahedral subunit. Biological subunit would include clockwise VP3. (D) Same as C, rotated x = 90°, y = 90°. (E) Similar to C with highlights (white) showing all RV-C15 surface amino acid residues conserved with >90% identity among all known RV-C isolates. See also Fig. S5.