Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease

Authors

  • Kenichi Morikawa,

    1. From the Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    Current affiliation:
    1. Division of Gastroenterology and Hepatology, Showa University School of Medicine, Tokyo, Japan
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Jérôme Gouttenoire,

    1. From the Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Céline Hernandez,

    1. Protein Analysis Facility, Lausanne, Switzerland
    2. Vital-IT Group, Swiss Institute of Bioinformatics, University of Lausanne, Switzerland
    Search for more papers by this author
  • Viet Loan Dao Thi,

    1. From the Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    Search for more papers by this author
  • Huong T.L. Tran,

    1. From the Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    Search for more papers by this author
  • Christian M. Lange,

    1. From the Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    Current affiliation:
    1. Department of Medicine I, University Hospital Frankfurt a.M., Germany
    Search for more papers by this author
  • Michael T. Dill,

    1. Division of Gastroenterology and Hepatology, University Hospital Basel, Switzerland
    Search for more papers by this author
  • Markus H. Heim,

    1. Division of Gastroenterology and Hepatology, University Hospital Basel, Switzerland
    Search for more papers by this author
  • Olivier Donzé,

    1. Adipogen, Epalinges, Switzerland
    Search for more papers by this author
  • François Penin,

    1. Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, UMR 5086, CNRS, University of Lyon, France
    Search for more papers by this author
  • Manfredo Quadroni,

    1. Protein Analysis Facility, Lausanne, Switzerland
    Search for more papers by this author
  • Darius Moradpour

    Corresponding author
    1. From the Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    • Address reprint requests to: Darius Moradpour, Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 44, CH-1011 Lausanne, Switzerland. E-mail: Darius.Moradpour@chuv.ch; fax: +41 21 314 47 18.

    Search for more papers by this author

  • Potential conflict of interest: Nothing to report.

  • Supported by grants 3100A0-122447 and 31003A-138484 from the Swiss National Science Foundation as well as by grant 09C53 from the Novartis Foundation.

Abstract

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. Conclusion: We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease. (Hepatology 2014;59:423–433)

Ancillary