Distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus C (HRV C)

Abstract

Background: Human rhinoviruses (HRVs) are the most frequently detected pathogens in acute respiratory tract infections (ARTIs) and yet little is known about the prevalence, recurrence, structure and clinical impact of individual members. During 2007, the complete coding sequences of six previously unknown and highly divergent HRV strains were reported. To catalogue the molecular and clinical features distinguishing the divergent HRV strains, we undertook, for the first time, in silico analyses of all available polyprotein sequences and performed retrospective reviews of the medical records of cases in which variants of the prototype strain, HRV-QPM, had been detected.

Methodology/principle findings: Genomic analyses revealed that the six divergent strains, residing within a clade we previously called HRV A2, had the shortest polyprotein of all picornaviruses investigated. Structure-based amino acid alignments identified conserved motifs shared among members of the genus Rhinovirus as well as substantive deletions and insertions unique to the divergent strains. Deletions mostly affected regions encoding proteins traditionally involved in antigenicity and serving as HRV and HEV receptor footprints. Because the HRV A2 strains cannot yet be cultured, we created homology models of predicted HRV-QPM structural proteins. In silico comparisons confirmed that HRV-QPM was most closely related to the major group HRVs. HRV-QPM was most frequently detected in infants with expiratory wheezing or persistent cough who had been admitted to hospital and required supplemental oxygen. It was the only virus detected in 65% of positive individuals. These observations contributed to an objective clinical impact ranging from mild to severe.

Conclusions: The divergent strains did not meet classification requirements for any existing species of the genus Rhinovirus or Enterovirus. HRV A2 strains should be partitioned into at least one new species, putatively called Human rhinovirus C, populated by members detected with high frequency, from individuals with respiratory symptoms requiring hospital admission.

Figure 1. Characterization of HRV A2 genomes.

(A) Scale representation of the coding region from prototype HRV A2 virus, HRV-QPM. Predicted protein products from the structural (VP4 to VP1) and non-structural (PRO-protease; POL-polymerase) regions are indicated. Regions are shaded alternatively for convenience and numbering indicates positions of predicted protease cleavage according to the start of the HRV-QPM polyprotein. Dashed lines indicate regions involved in species classifications. (B) HRV A2 amino acid similarity to HRV A (n = 34) and B (n = 13), or HEV (n = 71) strains as determined by SimPlot© analysis. Plots indicate the percentage similarity of a 50% consensus sequence from each species' polyprotein compared to the HRV A2 strains. Average similarity across the polyprotein is indicated by dotted line. Averaged values for the complete coding, P1 and 2C+3CD regions are indicated within the inset of each panel.

Figure 2. Comparison of predicted protomers.

(A) A simplified depiction of two protomers in opposition on a viral capsid (shaded areas, adapted from [37]). As a guide for visualising loop length, a dashed and a dotted line are spaced equidistantly and represent proximal and distal positions from the virion core, respectively. (B) Ribbon depiction of two opposing viral protomers from HRV-QPM, HRV-2 (minor group) and HRV-14 (major group). HRV-QPM proteins were predicted by in silico matching to the empirically derived HRV-16 and HRV-14 structure (materials and methods). Protomers consist of one copy each of VP1, VP2, VP3 and VP4. β-sheets are depicted as flat arrows and α-helices as coiled ribbons. The formation of VP1-VP3 into eight anti-parallel β-sheets is indicative of the ‘jellyroll’ conformation typical in picornaviruses. Major differences in the predicted HRV-QPM VP1 include the shortened external loops between β-sheets (asterisk) and an additional C-terminal sheet-loop-sheet formation (arrow indicates the same location on all protomers for comparison).

Figure 3. Predicted HRV-QPM pentamers compared to representative major (HRV-14) and minor (HRV-2) group HRV pentamers derived from crystallography data.

(A) HRV-QPM versus HRV-14 SimPlot data projected onto a space filling depiction of the predicted HRV-QPM pentamer. Shading represents the amino acid identity (26–69%). The yellow dashed triangle represents a single icosahedral asymmetric unit (T = p3 conformation) composed of VP1 and VP2 from the same protomer and VP3 for an adjacent protomer. The major group domains of interest are divided between two asymmetric units for ease of viewing. Receptor (white) and antigenic (red) sites are shown in outline. (B) Top view ribbon depiction of a major group HRV pentamer (HRV-14; gray) with labelled antigenic neutralisation sites (NImIA-III, green) and combined HRV A (HRV-16) and B (HRV-14)ICAM-1 receptor footprints (red) , . Magnified areas of interest (boxed) highlight computer-based predictive comparisons to the equivalent HRV-QPM (orange) structures of interest. Arrows indicate structures and corresponding sequences of interest (refer to text). (C) HRV-QPM versus HRV-2 SimPlot data projected onto the HRV-QPM pentamer. The domains of interest are mostly shown within a single asymmetric unit. (D) A minor group pentamer (HRV-2; gray) including antigenic sites (sites A–C, green) and VLDL-R footprint (red) . Attachment of the VLDL-R involves adjacent VP1 molecules. Magnified VP1 area represents one half of a VLDL-R footprint . Amino acid substitutions (arrowed) contributed to the differences between minor group sites B and C.

Figure 4. Comparison of HRV-QPM and HEV structures at the sites comprising HEV receptor footprints.

Figures indicate comparison of the predicted structures of HRV-QPM (orange) with representative HEVs (gray) in regions identified as HEV receptor footprints (red) across VP1, VP2 and VP3 proteins. RMSD values shown for conformational comparison of CAR and HRV-QPM structures, in angstroms.

Figure 5. Predicted virion capsid structures.

(A) Replication (×60) and mapping of the predicted HRV-QPM protomer onto a T = 1 icosahedral lattice (representing T = p3 configuration). (B) 3D rendering of predicted HRV capsids providing imagery similar to that obtained by cryo-electron microscopy reconstruction at 10 Å resolution. HRV-2, -14 and -16 predicted structures were derived from crystallography data. Each viral capsid has been depth cued to demonstrate canyon structure; yellow indicates surface detail and blue identifies areas of greatest depth. (C) Numbers superimposed over the lattice indicate the fold axis. The simplified position of the canyon is indicated by a circle on the capsid. All models were rendered and oriented identically, as determined by VIPERdb .

Figure 6. Neighbour-joining phylogeny based on representative full-length picornavirus polyprotein sequences.

Trees are unrooted and relevant nodes are labelled with bootstrap values (%) (see materials and methods for details). Species are indicated next to vertical bars. CV-Coxsackievirus A; EV-Echovirus; HEV-Enterovirus; HPV-Poliovirus.