title: NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.
abstract: Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.
authors.affiliation: Inserm U1019, CNRS UMR8204, Center for Infection & Immunity of Lille (CIIL), Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.
authors.forename: Costin-Ioan
authors.initials: CI
authors.lastname: Popescu
authors.forename: Nathalie
authors.initials: N
authors.lastname: Callens
authors.forename: Dave
authors.initials: D
authors.lastname: Trinel
authors.forename: Philippe
authors.initials: P
authors.lastname: Roingeard
authors.forename: Darius
authors.initials: D
authors.lastname: Moradpour
authors.forename: Véronique
authors.initials: V
authors.lastname: Descamps
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.forename: Laurent
authors.initials: L
authors.lastname: Héliot
authors.forename: Yves
authors.initials: Y
authors.lastname: Rouillé
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D014018:Tissue Distribution; D014758:Viral Core Proteins / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000235:genetics / Q000502:physiology*; D000086082:Viroporin Proteins
chemical_list: D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C069130:nucleocapsid protein, Hepatitis C virus; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins; C091015:glycoprotein E2, Hepatitis C virus