Insights into the genetic basis for natural phenotypic resistance of human rhinoviruses to pleconaril.

Recent phylogenetic analyses of the deduced amino acid sequence of the major viral capsid protein (VP1) of all human rhinovirus (HRV) serotypes revealed two distinct species within the genus: species A (75 serotypes) and species B (25 serotypes). Pleconaril is a novel capsid inhibitor of HRVs. All 75 species A serotypes and 18 of the 25 species B serotypes are susceptible to inhibition by pleconaril in cell culture. The seven resistant serotypes are HRV-4, -5, -42, -84, -93, -97 and -99. We were interested in understanding the genetic basis for phenotypic resistance to pleconaril among these naturally occurring viruses. We compared the 25 amino acids of VP1 that comprise the drug-binding pocket of susceptible and resistant species B viruses. A consistent difference was observed at two positions: the vast majority of susceptible viruses had tyrosine and valine at VP1 residues 152 and 191, respectively (Y(152) and V(191)); all resistant viruses had phenylalanine and leucine at these positions (F(152) and L(191)). HRV-14, a pleconaril susceptible virus, has a drug-binding pocket amino acid composition that differs from the naturally resistant HRV-5 and HRV-42 only at these two positions. To gain further insight into the role of these specific residues in natural resistance to pleconaril, we substituted the amino acids at these two positions individually and in combination in an infectious clone of HRV-14 and tested the rescued virus for susceptibility to pleconaril and virion stability. The results indicate that substitution of V(191) to Leu in HRV-14 has a profound negative impact on drug susceptibility but that full resistance to pleconaril is only seen when combined with Phe at position 152 in a HRV-14 double variant (F(152), L(191)). These data identify L(191) in species B HRV as a potentially key residue in conferring significantly reduced susceptibility to pleconaril. These results may be useful in distinguishing naturally occurring viral resistance to pleconaril from treatment-emergent resistance.

Structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds.

Pleconaril is a broad-spectrum antirhinovirus and antienterovirus compound that binds into a hydrophobic pocket within viral protein 1, stabilizing the capsid and resulting in the inhibition of cell attachment and RNA uncoating. When crystals of human rhinovirus 16 (HRV16) and HRV14 are incubated with pleconaril, drug occupancy in the binding pocket is lower than when pleconaril is introduced during assembly prior to crystallization. This effect is far more marked in HRV16 than in HRV14 and is more marked with pleconaril than with other compounds. These observations are consistent with virus yield inhibition studies and radiolabeled drug binding studies showing that the antiviral effect of pleconaril against HRV16 is greater on the infectivity of progeny virions than the parent input viruses. These data suggest that drug integration into the binding pocket during assembly, or at some other late stage in virus replication, may contribute to the antiviral activity of capsid binding compounds.

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

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.