Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C

Abstract

A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.

Figure 1. Propagation of HRV in mucosal organ cultures.

(a) Viral replication in single sinus organ culture infected with either high or low doses of HRV-A16. (b) Growth curve of HRV-A1 (triangles) and HRV-A16 (squares) strains in organ cultures after inoculation (1 × 10⁹ vRNA copies per ml) of sinus (solid lines) versus adenoidal (dashed lines) tissue (means ± s.d.). (c) Serial propagation (72 h) of a clinical isolate (HRV-A78) in three successive sinus organ cultures. (d) Propagation of HRV-C15 in sinus mucosal organ cultures. Cultured sinus tissue (passage 1) was inoculated with NLF containing HRV-C15, and serially passaged (passages 2–7), resulting in either high (≥2 × 10⁸ vRNA copies per ml) or low viral yields. (e) Growth curve of HRV-C15 (means, ± s.d.) in sinus organ cultures revealing distinct replication kinetics and viral yields. Organ cultures are designated according to tissue donors (dn).

Figure 2. HRV-C15 localization in sinus mucosa.

(a) Sinus cultures were inoculated with medium alone (left) or HRV-C15 (center and right), and whole mounts of the tissue were analyzed for HRV-C15 RNA by in situ hybridization (purple stain). Scale bars, 1 mm. (b) Higher magnification view of the areas boxed in panel a, showing uninfected cells (left) or cells containing viral RNA (center and right). Scale bars, 0.15 mm. (c) Sections of mock- (left) or HRV-C–infected (center and right) sinus tissue. Right image is counterstained with eosin (pink). Scale bars, 15 μm.

Figure 3. Neighbor-joining phylogenetic tree based on full-length nucleotide sequences of HRV-A, HRV-B and HRV-C.

Complete 5′ and 3′ UTR sequences and the first and second codon positions of the open reading frames were considered (MEGA 4.1 software). All major nodes are labeled with bootstrap values (% of 1,000 replicates). HRV-A and HRV-B reference strain accession numbers correspond to those published previously. The HRV-C15 (W10) genome sequenced in this study is shown in bold type. Branch lengths are proportional to nucleotide similarity (p distance). Human enteroviruses (HEV) are included as an outgroup. HRV-C types (followed by strain designations and accession numbers) correspond to the recent classification proposal.

Figure 4. HRV-C composition at known receptor footprint sites, binding characteristics and drug sensitivity.

(a) WebLogo depiction shows the dominant amino-acid compositions at alignment positions with ICAM-1 (HRV-B14 or HRV-A16) or LDLR (HRV-A2) footprint contact residues. Human coxsackievirus A21-only locations are not shown. Positions are labeled either by alignment rank (for example, 205, 206 and so on), or by the structural name of the virus protein residue contributing to the footprint (for example, 16-G-1-148). Compositions were tabulated separately for minor-group (14 strains) or major-group (119 strains) HRV-A and HRA-B and HRV-C (11 strains). Alignment positions identified as compositional matches (circled) or mismatches (all others) to the HRV-C by Pearson or Spearman statistical analyses for each receptor footprint cohort are shown. (b–d) Inhibition of virus attachment in HeLa cells (b), PBE cells (c) or sinus mucosa (d) using receptor-blocking antibodies. Viral RNA was quantified in cell lysates and normalized to β-actin (ACTB) expression (means ± s.d., n ≥ 3). *P < 0.05 versus medium control. (e) Inhibition of virus growth in sinus mucosa by WIN56291 (means ± s.d.).

Figure 5. RNA transcripts derived from pC15 clone are infectious.

(a) Cytopathic effects observed 24 h after transfection of WisL and HeLa cells with full-length HRV-A16 or HRV-C15 RNA. Scale bars, 100 μm. (b) Growth curve analysis of HRV-C15 progeny virus recovered after transfection of WisL cells. (c) Electron microscopy of concentrated cell lysates obtained 24 h after transfection of WisL cells with HRV-C15 RNA. Scale bar, 25 nm.