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.

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy.

Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.

Hepatitis C virus (HCV) is a positive-strand RNA virus that primarily infects human hepatocytes. Infections with HCV constitute a global health problem, with 180 million people currently chronically infected. Recent studies have reported that cholesterol 25-hydroxylase (CH25H) is expressed as an interferon-stimulated gene and mediates antiviral activities against different enveloped viruses through the production of 25-hydroxycholesterol (25HC). However, the intrinsic regulation of human CH25H (hCH25H) expression within the liver as well as its mechanistic effects on HCV infectivity remain elusive. In this study, we characterized the expression of hCH25H using liver biopsies and primary human hepatocytes. In addition, the antiviral properties of this protein and its enzymatic product, 25HC, were further characterized against HCV in tissue culture. Levels of hCH25H messenger RNA were significantly up-regulated both in HCV-positive liver biopsies and in HCV-infected primary human hepatocytes. The expression of hCH25H in primary human hepatocytes was primarily and transiently induced by type I interferon. Transient expression of hCH25H in human hepatoma cells restricted HCV infection in a genotype-independent manner. This inhibition required the enzymatic activity of CH25H. We observed an inhibition of viral membrane fusion during the entry process by 25HC, which was not due to a virucidal effect. Yet the primary effect by 25HC on HCV was at the level of RNA replication, which was observed using subgenomic replicons of two different genotypes. Further analysis using electron microscopy revealed that 25HC inhibited formation of the membranous web, the HCV replication factory, independent of RNA replication. Conclusion: Infection with HCV causes up-regulation of interferon-inducible CH25H in vivo, and its product, 25HC, restricts HCV primarily at the level of RNA replication by preventing formation of the viral replication factory. (HEPATOLOGY 2015;62:702-714)