Interaction of Hepatitis C Virus Protein Ns5A with Host Cofactor Cyclophilin A

Cyclophilin A (CyPA) and its peptidyl-prolyl cis/trans isomerase (PPIase) activity plays an essential role in hepatitis C virus (HCV) replication. Mounting evidence indicate nonstructural protein 5A (NS5A) of HCV as the major target of CyPA. We have identified a dipeptide region in a conserved region of NS5A domain 2 (D316/Y317) that regulates dependence of HCV upon CyPA and sensitivity to cyclophilin inhibitors. We have systematically characterized all proline residues in the C-terminal region of NS5A with functional replication assays, providing a high-resolution mapping of the relative contribution of each proline towards modulating CyPA sensitivity. Additionally, we have used biochemical binding assays to map the precise regions and residues within NS5A that regulate recruitment of CyPA. These data indicate a conserved tandem CyPA-binding site flanking either side of the D316/Y317 motif. Next, we have used NMR spectroscopy to examine the structural features surrounding this motif and the implications of mutations that relieve CyPA dependence. These data indicate the mutations at D316/Y317 induce structural changes in the region which are sensitive to disruption by CyPA. A promising inhibitor of CyPA is currently in phase II clinical trials for the interferon-free treatment of HCV. These data enhance our understanding of the role CyPA plays in the HCV life cycle and therefore further our understanding of mechanism by which clinical CyPA inhibitors suppress HCV infection in patients.