Essential Role of Cyclophilin A for Hepatitis C Virus Replication and Virus Production and Possible Link to Polyprotein Cleavage Kinetics

Abstract

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Increasing Rate of Cleavage at Boundary between Non-structural Proteins 4B and 5A Inhibits Replication of Hepatitis C Virus

“…Val and Iso) at the P3 and P4 positions (supplemental Fig. 2), consistent with what has also been observed at the NS5A5B boundary (31). From both the luciferase and colony-forming assays, we concluded that the P433 VV mutation, which gives rapid cleavage of the NS4B5A boundary, severely impairs replication.…”

Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

“…The power of this method is illustrated by NS5A and CypA. These two proteins are involved in HCV assembly (57,58); they cosediment with virions in density gradients (Figs. 1A and 2E), but they are not detectable in virus fractions after affinity purification (Fig.…”

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

“…In this respect, NS5A is of particular interest because it is the target of efficient direct acting antivirals (e.g. daclatasvir) (7) and its interaction with the human cyclophilin A (CypA), an essential peptidylprolyl cis-trans isomerase (PPIase) (8,9), is also targeted by the most advanced host-targeted antiviral (alisporivir) (10,11).…”