Inhibition of HCV Replication by Cyclophilin Antagonists Is Linked to Replication Fitness and Occurs by Inhibition of Membranous Web Formation

BACKGROUND & AIMS: Replication of hepatitis C virus (HCV) requires host cell factors, such as cyclophilin A (CypA). CypA binds to HCV's nonstructural protein (NS)5A to promote replication of viral RNA. CypA antagonists, such as cyclosporines, are potent inhibitors of HCV replication. NS2 modulates sensitivity of HCV to cyclosporines. We investigated why cyclosporines require NS2 to increase their inhibitory effect and how they block HCV replication. METHODS: We determined replication fitness and sensitivity of various HCV replicons, containing or lacking NS2, to cyclosporine and other direct-acting antiviral agents. We also analyzed the effects of cyclosporine on membranous web formation by electron microscopy. RESULTS: NS2-5B replicons of genotype 2a (JFH1), but not genotype 1b, had increased sensitivity to cyclosporine. This difference was lost with replication-attenuated NS3-5B JFH1 RNAs, showing that cyclosporine sensitivity is linked to reduced replication fitness of NS2-containing HCV RNAs. Fitness also determined sensitivity to a nucleoside analogue and an NS5A inhibitor, but not to telaprevir. Cyclosporine blocked de novo formation of the membranous web, but had little effect on established membranous replication factories. This block was prevented by cyclosporine resistance mutations in NS5A. CONCLUSIONS: Cleavage at the NS2/3 junction is a rate-limiting step in replication of particular HCV isolates and determines their sensitivity to CypA inhibitors. These drugs target de novo formation of the membranous web and RNA replication.