VP1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds

Abstract

Rhinoviruses are the most common infectious agents of humans. They are the principal etiologic agents of afebrile viral upper-respiratory-tract infections (the common cold). Human rhinoviruses (HRVs) comprise a genus within the family Picornaviridae. There are >100 serotypically distinct members of this genus. In order to better understand their phylogenetic relationship, the nucleotide sequence for the major surface protein of the virus capsid, VP1, was determined for all known HRV serotypes and one untyped isolate (HRV-Hanks). Phylogenetic analysis of deduced amino acid sequence data support previous studies subdividing the genus into two species containing all but one HRV serotype (HRV-87). Seventy-five HRV serotypes and HRV-Hanks belong to species HRV-A, and twenty-five HRV serotypes belong to species HRV-B. Located within VP1 is a hydrophobic pocket into which small-molecule antiviral compounds such as pleconaril bind and inhibit functions associated with the virus capsid. Analyses of the amino acids that constitute this pocket indicate that the sequence correlates strongly with virus susceptibility to pleconaril inhibition. Further, amino acid changes observed in reduced susceptibility variant viruses recovered from patients enrolled in clinical trials with pleconaril were distinct from those that confer natural phenotypic resistance to the drug. These observations suggest that it is possible to differentiate rhinoviruses naturally resistant to capsid function inhibitors from those that emerge from susceptible virus populations as a result of antiviral drug selection pressure based on sequence analysis of the drug-binding pocket.

FIG. 1.

Phylogenetic tree of HRV-A species members based on VP1 deduced amino acid sequence. The neighbor-joining dendrogram is based on a maximum-likelihood distance matrix calculated in PHYLIP (see Materials and Methods). The distance matrix was generated in PROTDIST, and the dendrogram was generated by using NEIGHBOR. The amino acids of the VP1 region were aligned in MegAlign by using the CLUSTAL W algorithm. Branch numbers represent bootstrap values for 100 trials, and arrows identify minor receptor-binding group viruses. To estimate branch lengths from the consensus tree, a distance matrix was generated by using PROTDIST from the original alignment of the VP1 region. Branch lengths for the user-defined consensus tree were estimated from the calculated distance matrix in FITCH. A single HRV-B species member (HRV-14) is included to provide perspective on the interspecies relationship within the genus. The branch length for HRV-14, which is 0.98, has been truncated for clarity.

FIG. 2.

Phylogenetic tree of HRV-B species members based on VP1 deduced amino acid sequence. The neighbor-joining dendrogram is based on a maximum-likelihood distance matrix calculated in PHYLIP (see Materials and Methods). The distance matrix was generated in PROTDIST, and the dendrogram was generated by using NEIGHBOR. The amino acids of the VP1 region were aligned in MegAlign by using the CLUSTAL W algorithm. Branch numbers represent bootstrap values for 100 trials. To estimate branch lengths from the consensus tree, a distance matrix was generated by using PROTDIST from the original alignment of the VP1 region. Branch lengths for the user-defined consensus tree were estimated from the calculated distance matrix in FITCH. A single HRV-A species member (HRV-16) is included to provide perspective on the interspecies relationship within the genus. The branch length for HRV-16, which is 1.07, has been truncated for clarity.

FIG. 3.

Interserotypic amino acid identity in HRV-A and HRV-B species members across VP1. The percent deduced amino acid sequence identity values were calculated by using the CLUSTAL W alignment algorithm and sequence distance option from MegAlign.

FIG. 4.

Stereo pair ribbon diagram of drug-binding pocket with bound pleconaril. The crystal structures of HRV-14 and HRV-16 are superimposed. Amino acid residues of HRV-16 are shown. The image was generated in MOE.

FIG. 5.

Relationship of amino acid conservation in drug-binding pocket and virus susceptibility to pleconaril inhibition. The mean fold difference (with 95% CI) in pleconaril EC₅₀ values between serotype pairs is plotted as a function of the number of amino acid differences in the drug-binding pocket.

FIG. 6.

Amino acid residues proposed to affect binding of central ring system of pleconaril and to affect natural resistance to the drug. The crystal structure of pleconaril bound in HRV-14 (red) and HRV-16 (blue) is shown. Two stereo pair images are shown to emphasize the difference in amino acid composition in the top of the virus pockets (upper image) and the position of the dimethylphenoxy group of pleconaril (bottom image). The image was generated in MOE.