Pharmacological disruption of hepatitis C NS5A protein intra- and intermolecular conformations

Non-structural 5A protein (NS5A) has emerged as an important pharmacological target for hepatitis C virus (HCV). However, little is known about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of virus replication. Here, we have presented two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A, and another that is inactive. Resistance to this antiviral was greater in genotype 1a than in genotype 1b replicons and mapped to domain 1 of NS5A. Using a novel cell-based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A, we showed that only the active antiviral specifically disrupted the close proximity of inter- and intramolecular positions of NS5A. The active antiviral, termed compound 1, caused a repositioning of both the N and C termini of NS5A, including disruption of the close approximation of the N termini of two different NS5A molecules in a multimolecular complex. These data provide the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the N termini of domain 1 in a NS5A complex but also alters the conformation of domain 3, and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals is needed to treat HCV infection, so a mechanistic understanding of what each component does to the viral machinery will be important.