title: HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors.
abstract: OBJECTIVE
  The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A.
  
  
  METHODS
  We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays.
  
  
  RESULTS
  We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A.
  
  
  CONCLUSIONS
  Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.
authors.affiliation: The Scripps Research Institute, La Jolla, USA.
authors.forename: Udayan
authors.initials: U
authors.lastname: Chatterji
authors.forename: Precious
authors.initials: P
authors.lastname: Lim
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bobardt
authors.forename: Stefan
authors.initials: S
authors.lastname: Wieland
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Cordek
authors.forename: Gregoire
authors.initials: G
authors.lastname: Vuagniaux
authors.forename: Francis
authors.initials: F
authors.lastname: Chisari
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
authors.forename: Paul
authors.initials: P
authors.lastname: Targett-Adams
authors.forename: Tanya
authors.initials: T
authors.lastname: Parkinson
authors.forename: Philippe A
authors.initials: PA
authors.lastname: Gallay
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology; D001665:Binding Sites; D021984:Cyclophilin A / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D016572:Cyclosporine / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000235:genetics; D005814:Genes, Viral; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D021984:Cyclophilin A; D016572:Cyclosporine; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
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