| Poliovirus is a member of the Picornaviridae family of viruses. Native poliovirus (N particle) is capable of undergoing structural rearrangement to an altered form (A particle) upon receptor binding in the course of cell entry. This conversion process is also known as uncoating. Uncoating is partially regulated by the presence of a hydrocarbon to the pocket of the VP1 subunit of the viral capsid. The precise chemical identity of this “pocket factor” is unknown. However antiviral compounds, which can inhibit the N to A transition, can also compete out the pocket factor for binding within VP1. While perfectly good vaccines exist against poliovirus, other members of the picornavirus family remain unchecked. For example, rhinovirus causes roughly 40% of all common colds, and non-polio enteroviruses cause about 15% of all upper respiratory tract infections. Recognizing a therapeutic need, we used poliovirus as a prototypical picornavirus to study the uncoating phenomenon from two different perspectives.; The first section of the thesis details methods for developing drugs to inhibit the uncoating process. Structure-based combinatorial libraries of pocket-binding, antiviral compounds were screened using a high-throughput mass spectroscopic assay. Positives were confirmed by traditional cell-based assay; a novel immunoprecipitation assay, which measured the rate constant of uncoating; and crystallographic analyses of virus-drug complexes. The process as a whole was highly successful, resulting in 8 positives from a 75 compound library. Three of the positives were micromolar or better inhibitors of either poliovirus or rhinovirus infectivity.; The second portion of the thesis involves using antiviral compounds as tools to determine the kinetic parameters of uncoating and the role of the pocket factor in regulating viral stability. We determined that the drugs entropically stabilize the virus from heat-induced uncoating. When the conversions were done in the presence of the viral receptor, the receptor was found to lower the activation energy of uncoating. The data presented here is the first strong evidence that viral receptors are catalysts. Moreover during receptor-mediated conversion in the presence of capsid-binding drugs, the enthalpic barrier for uncoating was increased, but the receptor countered this with a combination of both entropic and enthalpic effects. |
