title: Investigation of a role for lysine residues in non-structural proteins 2 and 2/3 of the hepatitis C virus for their degradation and virus assembly.
abstract: It has been demonstrated that both uncleaved, enzymitically inactive NS2/3 and cleaved NS2 proteins are rapidly degraded upon expression in cells, phenomena described to be blocked by the addition of proteasome inhibitors. As this degradation and its regulation potentially constitute an important strategy of the hepatitis C virus (HCV) to regulate the levels of its non-structural proteins, we further investigated the turnover of these proteins in relevant RNA replication systems. A lysine-mutagenesis approach was used in an effort to prevent protein degradation and determine any effect on various steps of the viral replication cycle. We show that, while NS2-lysine mutagenesis of protease-inactive NS2/3 results in a partial stabilization of this protein, the increased NS2/3 levels do not rescue the inability of NS2/3 protease inactive replicons to replicate, suggesting that uncleaved NS2/3 is unable to functionally replace NS3 in RNA replication. Furthermore, we show that the cleaved NS2 protein is rapidly degraded in several transient and stable RNA replicon systems and that NS2 from several different genotypes also has a short half-life, highlighting the potential importance of the regulation of NS2 levels for the viral life cycle. However, in contrast to uncleaved NS2/3, neither ubiquitin nor proteasomal degradation appear to be significantly involved in NS2 degradation. Finally, although NS2 lysine-to-arginine mutagenesis does not affect this protein's levels in a JFH-1 cell culture infection system, several of these residues are identified to be involved in virion assembly, further substantiating the importance of regions of this protein for production of infectious virus.
authors.affiliation: Goodman Cancer Centre and Department of Biochemistry, McGill University, Montréal, QC H3A 1A3, Canada.
authors.forename: Sarah
authors.initials: S
authors.lastname: Welbourn
authors.affiliation: Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Jirasko
authors.affiliation: Goodman Cancer Centre and Department of Biochemistry, McGill University, Montréal, QC H3A 1A3, Canada.
authors.forename: Valérie
authors.initials: V
authors.lastname: Breton
authors.affiliation: Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Reiss
authors.affiliation: Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS, Université de Lyon, IFR128 BioSciences Gerland-Lyon Sud, F-69367 Lyon Cedex 07, France.
authors.forename: Francois
authors.initials: F
authors.lastname: Penin
authors.affiliation: Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
authors.affiliation: Goodman Cancer Centre and Department of Biochemistry, McGill University, Montréal, QC H3A 1A3, Canada.
authors.forename: Arnim
authors.initials: A
authors.lastname: Pause
mesh_terms: D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D004279:DNA, Viral; D007976:Leupeptins; C097010:NS2 protein, Hepatitis C virus; D017361:Viral Nonstructural Proteins; D003546:Cysteine Endopeptidases; C107824:NS2-3 protease; D008239:Lysine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde