Characterization of poliovirus variants selected for resistance to the antiviral compound V-073

Abstract

V-073, a small-molecule capsid inhibitor originally developed for nonpolio enterovirus indications is considerably more potent against polioviruses. All poliovirus isolates tested to date (n = 45), including wild, vaccine, vaccine-derived, and laboratory strains, are susceptible to the antiviral capsid inhibitor V-073. We grew poliovirus in the presence of V-073 to allow for the identification of variants with reduced susceptibility to the drug. Sequence analysis of 160 independent resistant variants (80 isolates of poliovirus type 1, 40 isolates each of types 2 and 3) established that V-073 resistance involved a single amino acid change in either of two virus capsid proteins, VP1 (67 of 160 [42%]) or VP3 (93 of 160 [58%]). In resistant variants with a VP1 change, the majority (53 of 67 [79%]) exhibited a substitution of isoleucine at position 194 (equivalent position 192 in type 3) with either methionine or phenylalanine. Of those with a VP3 change, alanine at position 24 was replaced with valine in all variants (n = 93). The resistance phenotype was relatively stable upon passage of viruses in cell culture in the absence of drug. Single-step growth studies showed no substantial differences between drug-resistant variants and the virus stocks from which they were derived, while the resistant viruses were generally more thermally labile than the corresponding drug-susceptible parental viruses. These studies provide a foundation from which to build a greater understanding of resistance to antiviral compound V-073.

Fig 1

Single-step growth curves. Cultures of LLC-MK₂ cells were infected at an MOI of 10 with drug-susceptible parent virus and two drug-resistant variants for each poliovirus type (types 1, 2, and 3) described in Table 3. The amount of infectious virus present at various times postinfection was quantified by plaque assay. Solid circles, parental viruses; diamonds, VP1 I194 variants (VP1 I192 for type 3); triangles, VP3 A24 variants.

Fig 2

Thermal inactivation of virus infectivity. Drug-susceptible parental virus and two drug-resistant variants for each poliovirus type (types 1, 2, and 3) described in Table 3 were exposed to 46°C for various times, and the remaining infectivity determined by plaque assay. Solid circles, parental viruses; diamonds, VP1 I194 variants (VP1 I192 for type 3); triangles, VP3 A24 variants.

Fig 3

Location in the poliovirus type 2 structure of predominant amino acids substitutions that confer resistance to V-073, based on the crystallographic structure of V-073 bound to poliovirus type 2 (10). Superolateral cutaway view of electron density maps showing V073 bound within the hydrophobic pocket of VP1. Density mapping at 1 Å resolution and 2 Å radius representing the original parental strain (cyan) with a wireframe overlay (yellow) of the resistant virus. Amino acid differences are shown as a liquorice representation with parental (magenta) and variant (yellow). The final frame in the figure shows the position of V073 (white) relative to the overall pentamer structure with VP1 (blue), VP2 (yellow), and VP3 (red); VP4 is not visible in this representation.