Characterization of Hepatitis C Virus Subgenomic Replicon Resistance to Cyclosporine in Vitro

The current treatment for hepatitis C virus (HCV) consists of a combination therapy of alpha interferon (IFN-alpha) and ribivirin (RBV). Due to IFN resistance and side effects, new classes of drugs are needed to combat HCV infection. Cyclosporine A (CsA), an immunosuppressive and anti-inflammatory drug, has been shown to suppress HCV via a mechanism independent of the IFN pathway. In order to study the mechanism of CsA action on HCV, CsA resistant strains of HCV subgenomic replicon were selected and characterized. Here we report that different levels of resistance can be seen in different replicons and that different sets of mutations are associated with the different levels of resistance. Several different single cell clones with varying levels of CsA resistance contained mutations in the nonstructural protein 5B (NS5B), the HCV-encoded polymerase. When engineered into wildtype replicon these mutations were sufficient to confer a certain degree of resistance, but not to the original levels of selected replicons. Furthermore, these mutations, both individually and in groups, were able to rescue the lethal phenotype of a point mutation in NS5B (P540A) that has been previously implicated in the blockade of cyclophilins binding. These results demonstrate that CsA exerts selective pressure on the HCV genome despite being known to act on a cellular protein and identify a major target of CsA-mediated inhibition of HCV replication.