A Preliminary Analysis of the Differences in Cyclosporin A Resistance between Amino Acid Residues P538 and P540 of the Hepatitis C Polymerase

Alexandra Kuzmishin, The College of Wooster

Abstract

Hepatitis C virus (HCV) infection can cause severe liver damage and is the leading cause of liver transplants in the United States. The recommended pegylated IFN-α/ribivirin therapy is not effective against all HCV genotypes. Other therapeutics are in development, including non-immunosuppressive Cyclosporine A (CsA) analogs. CsA, prescribed to prevent transplant rejection, exhibits anti-HCV activity due to its ability to bind human cyclophilins. Cyclophilins, a subfamily of peptidyl prolyl isomerases, promote HCV RNA replication, in part by stimulating RNA binding by the HCV polymerase, NS5B. There is concern that some HCV genotypes are resistant to CsA and its analogs. To elucidate a) the mechanisms of resistance and b) the mechanism of CsA's anti-HCV activity, researchers have previously selected for CsA-resistant HCV replicons and have begun characterizing how these mutations alter virus-cyclophilin interactions. I aimed to extend these studies by analyzing a subset of NS5B mutations that contribute to CsA resistance and/or disrupt the NS5B-cyclophilin interaction. The I432V mutation confers resistance to CsA, whereas P540A is thought to disrupt the ability to bind Cyclophilin B (CyPB) and is lethal to the virus. Because I432V rescues the P540A mutant, researchers hypothesized that I432V/P540A would be resistant to CsA, but found that it was susceptible. This result suggests that P540A was previously mischaracterized and/or that CyPB binds another Proline (possibly P538). To address these possibilities, I expressed and purified the NS5B-I432V protein to compare to NS5B-P540A, NS5B-I432V/P540A, wildtype NS5B in RNA synthesis and binding assays. The final protein was 4.7 M and had few impurities; however, the enzyme assays were unsuccessful. I also engineered a plasmid for expression of NS5B-P538T for comparison to P540A. Due to trouble expressing P538T, this comparison was not accomplished.