title: NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication.
abstract: Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.
  
  
  OBJECTIVE
  Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Inés
authors.initials: I
authors.lastname: Romero-Brey
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Carola
authors.initials: C
authors.lastname: Berger
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kallis
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Androniki
authors.initials: A
authors.lastname: Kolovou
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Paul
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Lohmann
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany Ralf_Bartenschlager@med.uni-heidelberg.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000378:metabolism* / Q000821:virology; D004252:DNA Mutational Analysis; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020815:Polyproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D050505:Mutant Proteins; D020815:Polyproteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase