Mutations in the Hepatitis C Virus Polymerase that Increase RNA Binding Can Confer Resistance to Cyclosporine A

Hepatitis C virus (HCV) infection leads to acute and chronic liver diseases, and new classes of anti-HCV therapeutics are needed. Cyclosporine A (CsA) inhibits HCV replication and CsA derivatives that lack the immunosuppressive function are currently in clinical trials as candidate anti-HCV drugs. Here we characterize several independently derived HCV replicons with varying levels of CsA resistance due to mutations in nonstructural protein 513 (NS5B), the HCV-encoded polymerase. Mutant HCV replicons engineered with these mutations showed resistance to CsA. The mutations reside in two distinct patches in the polymerase: the template channel and one face of a concave surface behind the template channel. Mutant NS5B made by cells expressing the HCV replicon had increased ability to bind to RNA in the presence of CsA. Purified recombinant NS5B proteins containing the mutations were better at de novo initiated RNA synthesis than the wild-type control. Furthermore, the mutant proteins were able to bind RNA with approximate to 8-fold higher affinity. Last, mutation near the template channel alleviated the lethal phenotype of a mutation in the concave patch, P540A. This intramolecular compensation for the HCV replicase function by amino acid changes in different domains was further confirmed in an infectious cell culture-derived virus system. Conclusion: An increased level of CsA resistance is associated with distinct mutations in the NS5B gene that increase RNA binding in the presence of CsA, and the intramolecular communications between residues of the thumb and the C-terminal domains are important for HCV replicase function. (HEPATOLOGY 2009;50:25-33.)