Cadherin-Related Family Member 3, a Childhood Asthma Susceptibility Gene Product, Mediates Rhinovirus C Binding and Replication

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

Yury A. Bochkova,1, Kelly Wattersb, Shamaila Ashrafa,2, Theodor F. Griggsa, Mark K. Devriesa, Daniel J. Jacksona, Ann C. Palmenbergb,3, and James E. Gerna,c,3

aDepartment of Pediatrics and cDepartment of Medicine, University of Wisconsin–Madison, Madison, WI 53792; and bInstitute for Molecular Virology, University of Wisconsin–Madison, Madison, WI 53706

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved March 17, 2015 (received for review November 5, 2014) Members of rhinovirus C (RV-C) species are more likely to cause been a major obstacle to the study of virus-specific characteristics wheezing illnesses and asthma exacerbations compared with that could lead to effective antiviral strategies for this common other rhinoviruses. The cellular receptor for these viruses was and important respiratory pathogen. We now report that human heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3), a member of the cadherin-related family member 3 (CDHR3) enables the cells cadherin family of transmembrane proteins, mediates RV-C normally unsusceptible to RV-C infection to support both virus entry into host cells, and an asthma-related mutation in this gene binding and replication. A coding single nucleotide polymorphism is associated with enhanced viral binding and increased progeny (rs6967330, C529Y) was previously linked to greater cell-surface yields in vitro. expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared Results with wild-type CDHR3, cells transfected with the CDHR3-Y529 var- In Silico Identification of Candidate RV-C Receptors. To obtain a iant had about 10-fold increases in RV-C binding and progeny working list of potential cellular receptors, we measured gene yields. We developed a transduced HeLa cell line (HeLa-E8) stably expression on microarray chips (Human Gene 1.0 ST Array, expressing CDHR3-Y529 that supports RV-C propagation in vitro. Affymetrix) in two series of experiments involving cells that were Modeling of CDHR3 structure identified potential binding sites either susceptible or not susceptible to RV-C infection (Fig. 1 that could impact the virus surface in regions that are highly con- and Fig. S1). In one experimental series, susceptible cells in- served among all RV-C types. Our findings identify that the asthma cluded whole sinus mucosal tissue specimens (n = 5), epithelial susceptibility gene product CDHR3 mediates RV-C entry into host cell suspension from sinus tissue, and nasal epithelium obtained cells, and suggest that rs6967330 mutation could be a risk factor via brushing, and unsusceptible cells, including monolayers of for RV-C wheezing illnesses. primary undifferentiated epithelial cells and transformed cell lines (n = 5, including HeLa). In a second experimental series, rhinovirus C | CDHR3 | receptor we compared three pairs of undifferentiated (monolayers) and fully differentiated (ALI) sinus epithelial cell cultures. ore than 160 types of rhinoviruses (RVs) are classified into Mthree species (A, B, and C) of enteroviruses in the family Significance picornaviridae. Viruses belonging to the RV-C species were only discovered in 2006 (1, 2), some 50 y after the initial identification The rhinovirus C (RV-C) species was first identified in 2006 and of other RVs (3), because these viruses are not detectable by is a major cause of acute respiratory illnesses in children and – standard tissue-culture techniques (4 7). RV-Cs are of special hospitalizations for exacerbations of asthma. In this study, we clinical interest because they can cause more severe illnesses discovered that expression of human cadherin-related family requiring hospitalization in infants and children compared with member 3 (CDHR3), a transmembrane protein with yet un- the RV-A or RV-B, and are closely associated with acute exac- known biological function, enables RV-C binding and replica- – erbations of asthma (8 10). tion in normally unsusceptible host cells. Intriguingly, we

After multiple attempts to grow RV-C in established cell lines found that a coding SNP (rs6967330, C529Y) in CDHR3, pre- were unsuccessful, we described the use of organ culture of hu- viously linked to wheezing illnesses and hospitalizations for man sinus mucosa to propagate a few RV-C clinical isolates in

childhood asthma by genetic analysis, also mediates enhanced MICROBIOLOGY vitro (11). Subsequently, we and others documented RV-C RV-C binding and increased progeny yields in vitro. Finally, growth in cultures of epithelial cells isolated from airway tissue using structural modeling, we identified potential binding sites specimens that had then become redifferentiated under air–liquid in CDHR3 domains 1 and 2 interacting with viral capsid surface interface (ALI) conditions (12, 13). Notably, only fully differ- regions that are highly conserved among RV-C types. entiated ALI cultures supported RV-C replication, whereas undifferentiated airway epithelial cell monolayers did not. RV-C Author contributions: Y.A.B., A.C.P., and J.E.G. designed research; Y.A.B., K.W., S.A., can only be produced using reverse genetics, as the full-length T.F.G., M.K.D., and A.C.P. performed research; Y.A.B., K.W., D.J.J., A.C.P., and J.E.G. ana- viral RNA transcripts synthesized in vitro are infectious when lyzed data; and Y.A.B., A.C.P., and J.E.G. wrote the paper. transfected into human cell lines (for example, HeLa, WisL, and The authors declare no conflict of interest. HEK293T) normally not susceptible to viral infection (11). This article is a PNAS Direct Submission. With these tools, it was possible to show that the RV-C re- Data deposition: The data reported in this paper have been deposited in the Gene Ex- ceptor is distinct from the intercellular adhesion molecule 1 pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE61396). (ICAM-1) and low-density lipoprotein receptor (LDLR) family 1To whom correspondence should be addressed. Email: [email protected]. members used by the other species of RV (14, 15). However, the 2Present address: ATCC Microbiology Systems, Manassas, VA 20110. existing RV-C culture systems have relatively low throughput 3A.C.P. and J.E.G. contributed equally to this work. and are labor intensive. Failure to identify the cellular receptor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and the inability to propagate RV-C in convenient cell lines has 1073/pnas.1421178112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1421178112 PNAS | April 28, 2015 | vol. 112 | no. 17 | 5485–5490 Downloaded by guest on September 24, 2021 A Gene Ontology terms and second experiments, respectively. We then performed ad- Plasma membrane Receptor ditional filtering steps to narrow the candidate gene lists on the n = 66 genes n = 65 genes basis of available Gene Ontology information (membrane lo- calization, receptor activity) and expression levels of the known rhinovirus receptor genes (Fig. 1A and Table S1). We identified a total of 12 common genes (represented by 14 probe sets) Exp Common Exp Exp Common Exp encoding proteins localized to plasma membrane or with pre- #1 n = 25 #2 #2 n = 23 #1 dicted or functionally demonstrated receptor activity, including members of the human MHC class II, stomatin, guanine nucleotide-binding, type I cytokine, and atypical chemokine receptor Expression level: and cadherin protein families (Fig. 1B). RV-C susceptible > 7 log2 > RV-C unsusceptible Plasma membrane Receptor CDHR3 Expression in HeLa Cells Enables RV-C Binding and Replication. n = 38 genes n = 26 genes We next selected a subset of candidate genes [interleukin 5 receptor, alpha (IL5RA); chemokine (C-C motif) receptor-like 1 (CCRL1); cadherin-related family member 3 (CDHR3); low density lipoprotein receptor class A domain containing 1 (LDLRAD1); Exp Common Exp Exp Common Exp calponin homology domain containing 2 (CHDC2); membrane- #1 n = 11 #2 12 genes #1 n = 11 #2 14 probe sets spanning 4-domains, subfamily A, member 8 (MS4A8)] for functional validation (Fig. 1B and Fig. S2). We transfected HeLa cells with plasmid DNAs encoding the identified genes under control of the CMV promoter. The cells were then exposed to a reporter 3 Common 3 virus (RV-C15-GFP) engineered to express GFP during replication n = 8 (Fig. 2A) to facilitate the analysis. This reporter virus replicated well in susceptible ALI cultures of airway epithelial cells (Fig. S3). We repeatedly detected some GFP expression only in cells transfected with CDHR3-expressing cDNA (Fig. 2B). B log2 expression To confirm that this result was not unique to the GFP-expressing virus, we then infected CDHR3-expressing HeLa cells with wild- +3.7 +13.4 type RV-C15. Relative to untransfected control cells, we again observed enhanced virus binding and evidence of vigorous rep- n n RV-C unsusceptible ( = 8) RV-C susceptible ( = 10) lication (about 2-log increase in RV-C RNA compared with in- STOML3 put) (Fig. 2C). Therefore, transfection-induced expression of DNAAF1 CDHR3 was sufficient to convert HeLa cells normally unsusceptible KLHL6 to RV-C infection into targets that supported virus binding and CCRL1 subsequent replication. CERKL → ENPP3 Mutation in CDHR3 (Cys529 Tyr) Increases RV-C Binding and Progeny IL5RA Yields. CDHR3 is a related member of the cadherin family of transmembrane proteins. Typically, cadherins are involved in PROM1 homologous cell adhesion processes that are important for epi- CDHR3 thelial polarity, cell–cell interactions, and tissue differentiation HLA-DRA (16–18). Four alleles of CDHR3 gene are described, of which HLA-DRA one representing a single nucleotide polymorphism (G→A) that HLA-DRA converts residue cysteine to tyrosine at position 529 (Cys529→ RGS22 Tyr, rs6967330), was recently linked with a much greater risk of GNA14 asthma hospitalizations and severe exacerbations in young children in a genome-wide association study (19). It has been shown that this point mutation in cadherin-repeat domain 5 near a calcium binding site leads to a marked increase in cell surface PSE Sinus #3 Sinus #4 Sinus #7 Sinus #8 WisL WisL Sinus #2 Cell susp Sinus #6 Sinus #1 Sinus #3 Sinus #1 HeLa PBE Sinus #5 Sinus #2 Nasal epi NCI-H358 monolayers whole tissue ALI expression of the CDHR3 protein, presumably by altering the protein conformation. Fig. 1. Identification of candidate RV-C receptors by gene expression analysis. We engineered this single amino acid change into CDHR3 (A) Venn diagrams showing the common receptor candidate genes identified in cDNA, inserted the recombinant FLAG tag (DYKDDDDK) two series of microarray experiments after the filtering steps. The differentially between the signal sequence and domain 1 to facilitate detection 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 of CDHR3 surface expression, and then expressed it in HeLa map showing clustering analysis of expression profiles of the 12 differentially cells. Fluorescent microscopy and Western blot analysis con- expressed genes in primary cells, cell lines, and whole-tissue samples. Candidate firmed enhanced surface expression of Y529 (Fig. 2D), but also genes selected for validation of RV-C receptor activity are shown in red. Ex- similar levels of overall cellular expression of C529 (wild-type) pression intensity values were analyzed by hierarchical clustering of samples and Y529 (risk allele) variant (Fig. 2E). Notably, we found a and genes using the Euclidean distance metric with centroid linkage method higher (≥7-fold) number of cells expressing GFP after infection using ArrayStar software v4.0 (DNASTAR). Color bar represents gene expression with reporter C15 virus (Fig. 2F) and about 10-fold greater RV- intensity (log scale). See also Figs. S1 and S2 and Table S1. 2 C15 binding and subsequent progeny yield, relative to the wild- type CDHR3 (Fig. 2G). The FLAG tag did not affect virus By in silico analysis of expression profiles, we identified a total binding or replication (Fig. 2G). Parallel experiments with an- of 415 and 347 genes, preferentially expressed (>eightfold dif- other human cell line (HEK293T) again showed that CDHR3 ference, adjusted P < 0.05) in virus-susceptible cells in the first expression conferred susceptibility to RV-C infection (Fig. S4).

5486 | www.pnas.org/cgi/doi/10.1073/pnas.1421178112 Bochkov et al. Downloaded by guest on September 24, 2021 CDHR3 (Fig. 3A). The phenomenon is not unique to the C15 virus A RV-C15-GFP -LISSA / GPS- x2 3B type, because HeLa-E8 cells were also susceptible to C2 and C41 5’-UTR 1A 1B 1C 1D 2A 2B 2C 3A 3C 3D 3’-UTR infection (Fig. 3A). Quantitative RT-PCR (RT-qPCR), Western eGFP A(n) blot analysis, and flow cytometry confirmed higher CDHR3 mRNA (over 400-fold increase) and protein expression in RV-C15-GFP RV-C15 B C 107 transduced HeLa-E8 cells compared with parental HeLa (control) cells (Fig. 3 B and C). 6 10 The RV-C15 growth kinetics in HeLa-E8 monolayers was 105 overall similar to that of representative clinical isolates of A and B species showing continuous increase in viral RNA over the 104 time of infection (Fig. 3D). High expression levels of the ICAM- Virus titre (PFUe) Virus 103 1 receptor used by the A16 and B52 strains to enter host cells 2h 48h might be responsible for about 1-log higher levels of their – D E binding (2 h postinfection, hpi) and progeny yields (16 42 hpi), Control CDHR3-C529 CDHR3-Y529 compared with that of C15. Analysis of the binding properties of CDHR3 fluorescently labeled RV-C15 (RV-C15-APC) by flow cytometry

Control C529 Y529 consistently showed increased binding to HeLa-E8 (22.1% of kDa positively stained cells) compared with control HeLa (3.72%) 120 cells (Fig. 3E).

cytoplasm 100 80 An Extended Model of CDHR3 Structure. The CDHR3 protein sequence (885 aa) has a short signal peptide (19 aa) and six tan- 60 demly repeated cadherin domains (∼100 aa each), followed by 50 transmembrane (63 aa) and C-terminal cytoplasmic (150 aa) surface domains (Fig. 4A). The structure of CDHR3 has not yet been experimentally determined, but an approximation for domains 2–6, as modeled on the mouse N-cadherin ectodomain structure RV-C15-GFP RV-C15 F G 108 (PDB ID code 3Q2W), is described elsewhere (19). We extended this model to include CDHR3 domain 1 (Fig. S5), using I-Tasser 107 (20) and Robetta (21) algorithms to template the domain 1 and 106 2 sequences on multiple linked-domain cadherin analogs (e.g.,

105 PDB ID codes 1FF5A, 1Q5CA, and 2QVIA). The consensus output was joined to the domains 2–6 model 104

Virus titre (PFUe) Virus coordinates using the align function of PyMol (Molecular Graphics 3 10 2h 48h System, v1.6). Multiple iterations of this process, specifying a range of templates, did not alter the general configuration of Fig. 2. RV-C15 replication in HeLa cells transfected with CDHR3 cDNA. the final, full-domain structure (average RMSD: 1.632). The (A) Genome structure of the RV-C15 infectious clone expressing GFP. Two extended model (Fig. 4B) provides predictions of residues viral 2A protease cleavage sites permitting the release of GFP after poly- displayed on various repeat unit faces, including the Cys → protein translation are indicated. (B) GFP expression 48 h after C15-GFP in- 529 Tyr mutation at the domain 5–6 junction that defines the fection of HeLa cells transfected with wild-type CDHR3 for 48 h. (Scale bar, “ ” 100 μm.) Representative image of three independent experiments. (C) RV- risk allele. C15 binding (2 hpi) and replication (48 hpi) in HeLa cells transfected with wild-type CDHR3 (o), or Lipofectamine 2000 control (Δ) for 48 h (n = 5, data Docking the CDHR3 to a Two-Protomer Model of the RV-C15 Capsid. are means ± SD). (D) Fluorescent microscopy images of cells transfected with On cell surfaces or between cells, cadherins self-dimerize through → FLAG-tagged versions of CDHR3 wild-type (C529) or Cys529 Tyr variant (Y529). reciprocal domain 1 pairings mediated by cysteine, tryptophan, or Permeabilized (cytoplasm) or nonpermeabilized (surface) HeLa cells were hydrophobic interactions (22). There can be multiple glycosyla- stained with the α-CDHR3 or α-FLAG antibodies, respectively, to visualize tion sites dispersed along the ectodomain. The dimer format of μ intracellular or surface expression. (Scale bar, 100 m.) (E) Western blot CDHR3 is unknown, but there is a single tryptophan (Trp ) analysis of CDHR3 expression in HeLa cells transfected with wild-type (C ) 76 529 residue in domain 1. According to the prediction algorithms, or Cys529→Tyr variant (Y529) for 24 h and probed with α-CDHR3 polyclonal antibodies and α-tubulin antibodies (loading control). (F) GFP expression there is also a single putative asparagine-linked glycosylation site 48 h after C15-GFP infection of HeLa cells transfected with CDHR3 Cys →Tyr (Asn186) in domain 2. Trp76 and Asn186 position as solvent ex-

529 MICROBIOLOGY variant for 48 h. (Scale bar, 100 μm.) Representative image of three independent posed on opposite sides of their respective domains (Fig. 4B). experiments. (G) RV-C15 binding (2 hpi) and replication (48 hpi) in HeLa cells Assuming interactions similar to other picornaviruses, the RV-C → □ → transfected with CDHR3 Cys529 Tyr variant ( ), FLAG-tagged CDHR3 Cys529 Tyr are expected to access distal rather than proximal locations rel- (o), or Lipofectamine 2000 control (Δ)for48h(n = 5, data are means ± SD). See ative to the cellular membrane. also Figs. S3 and S4. To test the feasibility of virus–receptor interactions, we docked the CDHR3 domains 1, 2, and 3 to a two-protomer model of RV- Development and Characterization of Transduced HeLa Cell Line C15 capsid (23). Preliminary mutagenesis experiments suggested that Asn , but not Trp , was participating in virus binding; Stably Expressing CDHR3-Y Variant. To confirm RV-C multi- 186 86 529 therefore, both queried algorithms [Gramm-X (24) and HAD- cycle replication and enable large-scale virus propagation, we DOCK (25)] were given only two constraints: (i) CDHR3 Asn186 established (by lentiviral transduction) a human epithelial cell should be at or near the interface and (ii) the solvent exposed line HeLa-E8 stably expressing CDHR3-Y529. CDHR3 is expressed (outer) surface of the capsid proteins should be involved. The in these cells as a GFP-fusion protein that is cotranslationally preferred complexes docked the receptor across the adjacent pro- cleaved because of the presence of a 2A peptide derived from tomers, impacting the virus where capsid proteins VP1, -2, and -3 porcine teschovirus-1 to facilitate clonal selection of transduced abut in a fivefold to twofold orientation (Fig. 4 C and D). In this cells. Similarly to transfected cells, RV-C15 efficiently replicated orientation, Asn186 of domain 2 is buried in VP1 contacts and do- (about 2-log increase in viral RNA) in cells stably expressing main 1 extends across the twofold axis with VP1 and VP2 contacts,

Bochkov et al. PNAS | April 28, 2015 | vol. 112 | no. 17 | 5487 Downloaded by guest on September 24, 2021 ABmRNA Protein E Control HeLa - mock Control HeLa - RV-C15-APC 107 1000 0.02% 0.03% 3.72% 0% 105 105 100 CDHR3 106 104 4 10 10 Tubulin 105 RV-C15 1 103 103 RV-C41 RV-C2 0 RV-C15- APC 2 2 Relative expression 10 10 Virus titre (PFUe) Virus 104 HeLa HeLa- HeLa HeLa- 0 99.7% 0.24% 0 96.3% 0.01% 2h 24h control E8 control E8 01102 103 104 105 0 102 103 04 105 C 250K D HeLa-E8 - mock HeLa-E8 - RV-C15-APC 9 HeLa-control 10 0.01% 22.1% 5 0.02% 0.05% 200K HeLa-E8 10 105 108 150K 104 104 107

SSC-A 100K 106 RV-C15 103 103 RV-A16 50K 5 RV-C15- APC 10 RV-B52 102 102 0 titre (PFUe) Virus 0 0 104 0% 99.9% 0.08% 77.8% -103 0 103 104 105 2h 8h 16h 24h 42h 01102 103 104 105 0 102 103 04 105 CDHR3 CDHR3-GFP CDHR3-GFP

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.

whereas domain 3 is free of the virus and Trp86 is on a nondocked CDHR3 is a member of a cadherin family of transmembrane face (Fig. 4 D and E). proteins with a yet unknown biological function that is highly The RV-C as a species conserves <75% capsid protein iden- expressed in human lung tissue (26), bronchial epithelium (19), tity, but the RV-C15 structure model predicts a single shared and during mucociliary differentiation in human airway epithe- receptor (23). The modeled CHDR3 footprint covers nearly lial cells in vitro (27). Members of this family are responsible for every surface residue of RV-C15 that is conserved within the communication between identical cells through calcium-dependent species (Fig. 4E). Moreover, although modeled as a monomer interactions. Cadherins on the same cell surface can self-associate unit, this particular receptor orientation would easily accom- into cis-dimers (lateral dimers) and cell adhesion is based on in- modate domain 1-mediated dimer contacts across the twofold teractions between identical cadherins on neighboring cell surfaces axis of the virus, or even other multimers around the fivefold. to form transdimers (28). Our structure modeling of the CDHR3- virus complex identified a potential binding site for monomers and Discussion dimers in a region of the RV-C15 capsid that is highly conserved The cure for the common cold has been elusive, in part because among different RV-C types. The docking exercise further suggests of incomplete information on the molecular biology of RV-C, that receptor domains 1 and 2 may be key to virus interactions, and which is an important cause of both upper and lower respiratory that glycosylation, particularly at Asn186, may be contributory. Such illnesses as well as acute exacerbations of chronic childhood models of proteins and their interactions will help guide studies asthma. Toward this goal, we performed genome-wide gene- using mutational analysis to accurately map virus binding domains expression analysis of cells that were either susceptible or not in CDHR3. susceptible to RV-C infection, selected and functionally vali- Notably, our findings demonstrate that a coding SNP (rs6967330) in CDHR3, which was previously associated with wheezing illnesses dated a subset of candidate receptor genes by expression in and hospitalizations in childhood asthma by genetic analysis (19), HeLa cells followed by infection with GFP-expressing RV-C15, is also associated with increased RV-C binding and progeny and established that human CDHR3 confers susceptibility to yields in vitro. If the same relationship is true in vivo, rs6967330 RV-C infection to normally unsusceptible cells. Furthermore, and the surface overexpression of CDHR3 could also be a spe- replication of multiple RV-C types was demonstrated in HeLa- cific risk factor for more severe RV-C illnesses. Another point to E8 cells with stable expression of CDHR3-Y529. Finally, we consider is that RV wheezing illnesses in infancy are a strong risk modeled the complete CDHR3 structure, and identified a pu- factor for subsequent childhood asthma and decreased lung tative docking site for CDHR3 monomers and dimers to bind function (29–31), but whether the viral infections serve a causal virus in a region with multiple residues conserved among all RV-C role is unknown (32). These findings suggest the possibility that types. These findings have great potential utility for RV re- the rs6967330 SNP could promote RV-C wheezing illnesses in search. Cell lines such as HeLa-E8 will enable production of infancy, which could in turn adversely affect the developing lung large quantities of virus that can be used to develop RV-C to increase the risk of asthma. vaccines and to determine the actual, rather than modeled, vi- Expression of CDHR3 increased RV-C binding and enabled rion crystal structure. In addition, the HeLa-E8 cells could be replication in normally unsusceptible host cells. These findings used to develop RV-C infectivity assays for antiviral drug suggest that CDHR3 is a functional receptor for RV-C. One screening, for detection of neutralizing antibody response to limitation of our results is that anti-CDHR3 antibodies to RV-C in clinical studies, and to grow RV-C clinical isolates extracellular domains or recombinant CDHR3 (such as the from samples of respiratory fluids. CDHR3–Fc fusion protein) are not currently available to test for

5488 | www.pnas.org/cgi/doi/10.1073/pnas.1421178112 Bochkov et al. Downloaded by guest on September 24, 2021 A

BCD

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.

direct interaction between the virus and putative receptor, which described previously (11). The protocol was approved by the University of will be necessary to further prove its receptor function. Another Wisconsin–Madison Human Subjects Committee. Primary airway epithelial point to consider is that our studies show some binding of RV-C cells were cultured submerged (undifferentiated monolayers) or at the ALI to control HeLa cells. This low-level binding could be non- (fully differentiated), as previously described (13, 40). specific, or could indicate that CDHR3 is expressed at a very low Viruses and Infection. Recombinant RV-C15, RV-C15-GFP, RV-C2, RV-C41, RV- level in control HeLa cells. Alternatively, there could be a A16, and RV-B52 were produced by transfecting viral RNA synthesized in vitro coreceptor for RV-C that interacts with CDHR3, or a cofactor from linearized infectious cDNA clones into WisL cells, as previously described that facilitates binding and entry. For example, bacterial surface (11, 40). Cells grown in 12-well plates (monolayers) or in Transwell polycarbonate polysaccharides stabilize polioviruses and facilitate cell attach- inserts (0.4-μm pore size; Corning) (ALI cultures) were inoculated with RV at ment by increasing binding to the viral receptor (33, 34). In any 106 PFUe per well or insert (unless other dose indicated), incubated for 2–4h case, experiments in our laboratory and elsewhere demonstrated at 34 °C, and washed three times with PBS to remove unattached input virus. that the RV-C interaction with native HeLa cells or other cell lines (e.g., A549) is not sufficient for efficient virus entry and RNA Extraction and RT-qPCR. Total RNA was extracted from sinus tissue or replication (5, 6, 35). cultured cells using the RNeasy Mini kit (Qiagen). Viral RNA concentrations were determined by RT-qPCR using Power SYBR Green PCR mix (Life Tech- MICROBIOLOGY In conclusion, no vaccines or effective antivirals are currently nologies), as previously described (11). Relative expression of CDHR3 mRNA available for RV (36, 37). Blocking interactions between major was determined by RT-qPCR using the CDHR3-qf and CDHR3-qr primers receptor group RVs and ICAM-1 inhibits infection, airway in- (Table S2). flammation, and illness (38, 39), but is ineffective against replication of minor receptor group viruses or RV-C types. Anti-CDHR3 an- Gene Expression Analysis. Total RNA samples were treated with RQ1 DNase, tibodies, soluble CDHR3, or short peptides specifically targeting labeled, fragmented and hybridized to Human Gene 1.0 ST arrays (Affy- virus-binding sites could serve as inhibitors of RV-C infection and metrix). Raw data (.cel files) were uploaded into ArrayStar software v4.0 provide a new therapeutic approach for RV-C–induced illnesses. (DNASTAR) for normalization and statistical analysis. Gene-expression data Development of RV-C–specific antiviral drugs may be especially have been deposited to Gene Expression Omnibus (GEO) database under accession no. GSE61396. important for treatment of infants and children with the rs6967330 asthma risk allele in CDHR3. Expression Plasmids and Transfection. Plasmids for expression of receptor candidate genes CCRL1 (Missouri S&T cDNA Resource Center), MS4A8 Materials and Methods (OriGene), CHDC2,andCDHR3 (TransOmic) were purchased. IL5RA and Expanded methods are presented in SI Materials and Methods. LDLRAD1 ORFs were PCR-amplified from a cDNA sample obtained from differentiated airway epithelial cells using the corresponding primers

Cell Cultures. Sinus epithelial tissue samples were obtained from residual (Table S2). The mutation in domain 5 (C529Y) of CDHR3 was engineered by surgical specimens from individuals undergoing sinus surgery and cultured as two-step PCR using the flanking (CDHR3-f3 and CDHR3-r3) and internal

Bochkov et al. PNAS | April 28, 2015 | vol. 112 | no. 17 | 5489 Downloaded by guest on September 24, 2021 (CDHR3-C529Y-f and CDHR3-C529Y-r) primers. The plasmid DNA was pre- Lipofectamine 2000 (Life Technologies) to produce lentivirus particles. HeLa pared by Plasmid Maxi kit (Qiagen) and transfected into monolayers of HeLa cells were transduced, selected with blasticidin (5 μg/mL) and cloned by or HEK293T cells using Lipofectamine 2000 (Life Technologies) according to limiting dilution in 96-well plates. The HeLa-E8 clone, showing the highest the manufacturer’s instructions. RV-C replication levels (over 2-log), was selected for further experiments.

Fluorescent Microscopy. HeLa cells plated on glass coverslips were transfected Flow Cytometry. Control or transduced cells grown in suspension were with 1 μg of pCDHR3-FLAG DNA using Lipofectamine 2000 (Life Technolo- washed, stained with Ghost 780 (Tonbo) exclusion dye, fixed, and per- gies) and fixed 24 h posttransfection. For detection of cell surface expression meabilized. Cells were then blocked [10% (vol/vol) FBS, 0.05% Tween-20 of CDHR3, nonpermeabilized fixed cells were washed (two times) with PBS, in PBS], washed, and reacted with anti-CDHR3 mAbs (Abcam, ab56549). blocked, and reacted with rabbit monoclonal anti-FLAG primary antibody After wash (three times) with PBS, cells were reacted with Alexa Fluor (Sigma, F2555). Cells were then washed (three times) and treated with Alexa 647-conjugated donkey anti-mouse secondary antibody (Life Technolo- Fluor 594 anti-rabbit antibody (Life Technologies). Next, for detection of gies), washed again (three times), and analyzed by flow cytometry. total cellular CDHR3 expression, cells were permeabilized, washed (three times), reblocked, and stained with rabbit polyclonal anti-CDHR3 (Sigma, Fluorescent Labeling of RV-C15 and Virus Binding Assay. The purified C15 virus HPA011218). After wash (three times) with PBS, cells were treated with was labeled with NHS ester fluorescent probe DyLight 650 (Thermo Scientific) Alexa Fluor 488 anti-rabbit antibodies (Life Technologies). following the manufacturer’s recommendations. Unincorporated dye was removed by fluorescent dye removal columns (Thermo Scientific). Monolayers Generation of Stable HeLa Cell Line Expressing CDHR3. The mutation in domain of HeLa cells were trypsinized, incubated with labeled virus (multiplicity of infection of 10) for 1.5 h at 25 °C, washed (three times) before fixation 5(C529Y) of CDHR3 was engineered in lentiviral vector pLX304 containing with 2% (wt/vol) paraformaldehyde, and analyzed by flow cytometry. wild-type CDHR3 sequence (TransOmic) by subcloning from pCDHR3-C529Y. We then added a 2A peptide sequence derived from porcine teschovirus-1 (41) and the GFP sequence to the 3′-end of CDHR3 using synthetic gene ACKNOWLEDGMENTS. We thank Shakher Sijapati, Marchel Hill, Maria – fragments (gBlocks, Integrated DNA Technologies) to encode the CDHR3– Leighton, and Rebecca Brockman-Schneider (University of Wisconsin Madison) for their technical assistance, and Alar Aab (University of Tartu) for advice GFP fusion protein, which is cotranslationally cleaved to facilitate clonal regarding the virus-labeling procedure. This study was supported by the Na- selection of transduced cells by direct fluorescent microscopy. The resulting tional Institutes of Health–National Institute of Allergy and Infectious Dis- plasmid, pLX304-CDHR3-C529Y-NPGP-GFP, was cotransfected with the mix- eases Grant U19-AI0104317 and by the National Institutes of Health–National ture of packaging plasmids (psPAX2 and pMD2.G) into the 293T cells using Heart, Lung, and Blood Institute Grant P01 HL070831.

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