Alisporivir is the most advanced host-targeting
antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV)
viremia in infected patients. In an attempt to further elucidate the mechanism of action of
alisporivir, HCV replicons resistant to the
drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of
alisporivir or in the absence of the
alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA,
RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from
alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to
alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that
alisporivir is associated with a low potential for development of viral resistance.