Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.
We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.
Hanoulle
Xavier
X
Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, IFR 147, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France.
Badillo
Aurélie
A
Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, Université de Lyon, IFR 128, BioSciences Gerland-Lyon Sud, F-69397 Lyon, France.
Wieruszeski
Jean-Michel
JM
Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, IFR 147, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France.
Verdegem
Dries
D
Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, IFR 147, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France.
Landrieu
Isabelle
I
Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, IFR 147, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France.
Bartenschlager
Ralf
R
Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
Penin
François
F
Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, Université de Lyon, IFR 128, BioSciences Gerland-Lyon Sud, F-69397 Lyon, France.
Lippens
Guy
G
Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, IFR 147, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France. Electronic address: guy.lippens@univ-lille1.fr.
D021984:Cyclophilin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021983:Cyclophilins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
D021984:Cyclophilin A; D021983:Cyclophilins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C081786:cyclophilin B; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase