title: Hepatitis C virus variants resistant to macrocyclic NS3-4A inhibitors subvert IFN-β induction by efficient MAVS cleavage.
abstract: OBJECTIVE
  The hepatitis C virus (HCV) NS3-4A protease is essential for the HCV life cycle and a prime target of antiviral treatment strategies. Protease inhibitors, however, are limited by emergence of resistance-associated amino acid variants (RAVs). The capacity to cleave and inactivate mitochondrial antiviral-signaling protein (MAVS) in the RIG-I-signaling pathway is a cardinal feature of NS3-4A, by which HCV blocks induction of interferon-(IFN)-β, thereby promoting viral persistence. Here, we aimed to investigate the impact of NS3-4A RAVs on MAVS cleavage.
  
  
  METHODS
  The impact of NS3-4A RAVs on MAVS cleavage was assessed using immunoblot analyses, luciferase reporter assays and molecular dynamics simulations to study the underlying molecular principles. IFN-β was quantified in serum from patients with different NS3-4A RAVs.
  
  
  RESULTS
  We show that macrocyclic NS3-4A RAVS with substitutions at residue D168 of the protease result in an increased capacity of NS3-4A to cleave MAVS and suppress IFN-β induction compared with a comprehensive panel of RAVs and wild type HCV. Mechanistically, we show the reconstitution of a tight network of electrostatic interactions between protease and the peptide substrate that allows much stronger binding of MAVS to D168 RAVs than to the wild-type protease. Accordingly, we could show IFN-β serum levels to be lower in patients with treatment failure due to the selection of D168 variants compared to R155 RAVs.
  
  
  CONCLUSIONS
  Our data constitutes a proof of concept that the selection of RAVs against specific classes of direct antivirals can lead to the predominance of viral variants with possibly adverse pathogenic characteristics.
authors.affiliation: Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany; Max-Planck-Institute for Informatics, Computational Biology & Applied Algorithmics, D-66123 Saarbrücken, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Welsch
authors.affiliation: Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Haselow
authors.affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gouttenoire
authors.affiliation: Technical University Munich, Center for Integrated Protein Science Munich (CIPS(M)) and Department of Life Science, D-85354 Freising-Weihenstephan, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Schneider
authors.affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Morikawa
authors.affiliation: Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Martinez
authors.affiliation: Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Susser
authors.affiliation: Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Sarrazin
authors.affiliation: Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zeuzem
authors.affiliation: Technical University Munich, Center for Integrated Protein Science Munich (CIPS(M)) and Department of Life Science, D-85354 Freising-Weihenstephan, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Antes
authors.affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland.
authors.forename: Darius
authors.initials: D
authors.lastname: Moradpour
authors.affiliation: Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany. Electronic address: Christian.Lange@kgu.de.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Lange
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000998:Antiviral Agents / Q000494:pharmacology*; D018929:Cell Culture Techniques; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D024882:Drug Resistance, Viral / Q000276:immunology; D005838:Genotype; D016174:Hepacivirus / Q000472:pathogenicity / Q000502:physiology; D019698:Hepatitis C, Chronic / Q000276:immunology / Q000378:metabolism / Q000821:virology; D006801:Humans; D016899:Interferon-beta / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism; D012367:RNA, Viral; D011971:Receptors, Immunologic; D012697:Serine Endopeptidases / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D000085242:Viral Proteases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000998:Antiviral Agents; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D016899:Interferon-beta; D020365:RNA Helicases; D012367:RNA, Viral; D011971:Receptors, Immunologic; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C505371:MAVS protein, human; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D000085242:Viral Proteases; C452820:RIGI protein, human
keywords: All-oral therapy; Cardif; Direct-acting antiviral agent; Innate immunity; RIG-I signaling