title: Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A.
abstract: OBJECTIVE
  Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response.
  
  
  METHODS
  In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways.
  
  
  RESULTS
  Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2.
  
  
  CONCLUSIONS
  Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner.
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Marie-Sophie
authors.initials: MS
authors.lastname: Hiet
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Bauhofer
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Margarita
authors.initials: M
authors.lastname: Zayas
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Hanna
authors.initials: H
authors.lastname: Roth
authors.affiliation: Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schirmacher
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Joschka
authors.initials: J
authors.lastname: Willemsen
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Grünvogel
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Bender
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Binder
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Lohmann
authors.affiliation: CIRI U1111/UMR5308, Tour Inserm-CERVI, Lyon, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Lotteau
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany. Electronic address: Alessia_Ruggieri@med.uni-heidelberg.de.
authors.forename: Alessia
authors.initials: A
authors.lastname: Ruggieri
authors.affiliation: Department for Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany. Electronic address: Ralf_Bartenschlager@med.uni-heidelberg.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000235:genetics*; D004195:Disease Models, Animal; D005260:Female; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy* / Q000473:pathology / Q000821:virology; D022781:Hepatocytes; D006801:Humans; D016898:Interferon-alpha / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D016898:Interferon-alpha; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: HCV; HCV-mediated MDA5 activation; NS5A; Subversion of interferon response