title: A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication.
abstract: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro(314)-Trp(316) turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro(314) of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.
authors.affiliation: From the CNRS, Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576, Université Lille1, F-59655 Villeneuve d'Ascq, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Dujardin
authors.affiliation: the Department of Infectious Diseases, Molecular Virology, University of Heidelberg, D-69120, Heidelberg, Germany, and.
authors.forename: Vanesa
authors.initials: V
authors.lastname: Madan
authors.affiliation: CNRS, Bases Moléculaires et Structurales des Systèmes Infectieux, IBCP, LabEx Ecofect, UMR 5086 and Université Lyon 1, F-69367, Lyon, France.
authors.forename: Roland
authors.initials: R
authors.lastname: Montserret
authors.affiliation: From the CNRS, Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576, Université Lille1, F-59655 Villeneuve d'Ascq, France.
authors.forename: Puneet
authors.initials: P
authors.lastname: Ahuja
authors.affiliation: From the CNRS, Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576, Université Lille1, F-59655 Villeneuve d'Ascq, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Huvent
authors.affiliation: From the CNRS, Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576, Université Lille1, F-59655 Villeneuve d'Ascq, France.
authors.forename: Helene
authors.initials: H
authors.lastname: Launay
authors.affiliation: the EA4529 and InstruL2, UFR de Pharmacie, Université Paris-Sud, F-92296 Châtenay-Malabry, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Leroy
authors.affiliation: the Department of Infectious Diseases, Molecular Virology, University of Heidelberg, D-69120, Heidelberg, Germany, and.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
authors.affiliation: CNRS, Bases Moléculaires et Structurales des Systèmes Infectieux, IBCP, LabEx Ecofect, UMR 5086 and Université Lyon 1, F-69367, Lyon, France.
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.affiliation: From the CNRS, Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576, Université Lille1, F-59655 Villeneuve d'Ascq, France.
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
authors.affiliation: From the CNRS, Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576, Université Lille1, F-59655 Villeneuve d'Ascq, France, xavier.hanoulle@univ-lille1.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Hanoulle
mesh_terms: D020816:Amino Acid Motifs; D021984:Cyclophilin A / Q000737:chemistry; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry; D008958:Models, Molecular; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D021984:Cyclophilin A; D064267:Intrinsically Disordered Proteins; D011392:Proline; D012367:RNA, Viral; D014364:Tryptophan; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: RNA replication; cyclophilin; hepatitis C virus (HCV); intrinsically disordered protein; nuclear magnetic resonance (NMR); prolyl isomerase; protein motif; viral protein