Human Rhinovirus B14 with Non-functional Drug-binding Pockets Recovers Infectivity through Stereochemically Constrained Mutations that Restore Capsid Flexibility

Human rhinovirus (RV) is one of the most frequent human pathogens. As causative agents of most common colds, RVs originate serious socioeconomic problems, and they are also associated to or exacerbate severe respiratory diseases. No anti-rhinoviral drugs or vaccines are available yet. In this study, amino acid residues that delimit a druggable pocket in the RV capsid were individually replaced by larger residues, which in most cases resulted in impaired virus infectivity. After serial infections of human host cells, the hampered mutant viruses invariably recovered full infectivity. Sequencing of the viral progeny revealed that, contrary to what has been observed when deleterious mutations were introduced in other virus capsid regions, those mutant RVs did not regain infectivity through the fixation of second-site mutations. Instead, infectivity was always recovered through reversion or pseudo-reversion events that replaced the deleterious bulky residue with a smaller residue, even though this strategy required, in some cases, the improbable fixation of two nucleotides in the corresponding codon. All-atom molecular dynamics simulations and determination of mechanical elasticity by atomic force microscopy revealed that the small-to-large, deleterious mutations reduced the capsid conformational flexibility; and the large-to-small, infectivity-restoring (pseudo)reversions led to recovery of the capsid flexibility. The stereochemistry of this druggable hydrophobic pocket in RV appears to be biologically constrained: The deleterious effect of reducing pocket volume and capsid flexibility is reverted during virus propagation only through same-site mutations that restore pocket volume and capsid flexibility. These constraints may help the further development of anti-RV drugs.