Identification and Characterization of Tandem Cyclophilin a Binding Sites in Non-Structural Protein 5A

Hepatitis C virus (HCV) is an enveloped RNA virus that often manifests into chronic infections of the liver, which represents a threat to human health due to the morbidity it presents within infected patients. The virus is especially persistent as current antiviral treatments have difficulty in restricting the infections, allowing most new cases of viral hepatitis to develop into persistent infections. It has emerged as a major causative agent of liver diseases, resulting in cirrhosis, liver failure, and hepatocellular carcinoma as the disease progresses with time. There is no prophylactic vaccine currently available to protect against HCV, nor is there an effective therapy capable of generating a sustained virologic response. HCV has shown the capability of developing resistance to antiviral compounds that target specific viral enzymes necessary for replication. HCV is found as quasispecies within an individual due to its error prone RNA-dependent RNA polymerase. The genetic heterogeneity of HCV allows it to be capable of escaping the effect of compounds that specifically target viral proteins. Several cellular cofactors have been identified recently that permit HCV to be infectious. The interactions of these cofactors with viral proteins shed light on the life cycle of HCV and warrant further study as potential cellular targets to restrict viral infectivity. One such cofactor is cyclophilin A (CyPA) which has peptidylproline isomerase (PPIase) activity. HCV strains with a reduced dependency on CyPA for replication were selected in a CyPA-knockdown cell line. Sequencing of these isolates revealed mutations within a dipeptide motif of domain 2 of NS5A for all clones (D316E and/or Y317N). Analysis of these phenotypes upon insertion into the full-length viral genome revealed the double mutant, termed DEYN, to most efficiently restore the infectivity of the virus in CyPA-knockdown cells to that of control cells. We have hypothesized several possible mechanisms for the reduced dependency on CyPA of DEYN virus. These mutations could potentially affect viral replication via the PPIase activity of CyPA which catalyzes the cis-trans isomerization of proline residues. Presumably, HCV requires CyPA as a host cofactor to isomerize one or more proline residues within NS5A, converting it from a non-functional, unfolded protein into a functional, folded conformer. The work described here identifies structural differences between wt and mutant NS5A sequences, identifies CyPA binding sites within domain II and LCS II, and describes critical determinants within CyPA and NS5A that permit their interactions. Furthermore, this work identified AphiPXW and [AP]LPP as CyPA binding motifs among the 7 genotypes tested.