title: Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A.
abstract: Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an α-helix. NMR analysis identifies two putative α-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.
authors.affiliation: Université Lille Nord de France, F-59000 Lille, France.
authors.forename: Dries
authors.initials: D
authors.lastname: Verdegem
authors.forename: Aurélie
authors.initials: A
authors.lastname: Badillo
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Wieruszeski
authors.forename: Isabelle
authors.initials: I
authors.lastname: Landrieu
authors.forename: Arnaud
authors.initials: A
authors.lastname: Leroy
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
authors.forename: Xavier
authors.initials: X
authors.lastname: Hanoulle
mesh_terms: D021984:Cyclophilin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010449:Peptide Mapping / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase
chemical_list: D021984:Cyclophilin A; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase