title: Inhibition of HCV translation by disrupting the structure and interactions of the viral CRE and 3' X-tail.
abstract: A phylogenetically conserved RNA structure within the NS5B coding region of hepatitis C virus functions as a cis-replicating element (CRE). Integrity of this CRE, designated SL9266 (alternatively 5BSL3.2), is critical for genome replication. SL9266 forms the core of an extended pseudoknot, designated SL9266/PK, involving long distance RNA-RNA interactions between unpaired loops of SL9266 and distal regions of the genome. Previous studies demonstrated that SL9266/PK is dynamic, with 'open' and 'closed' conformations predicted to have distinct functions during virus replication. Using a combination of site-directed mutagenesis and locked nucleic acids (LNA) complementary to defined domains of SL9266 and its interacting regions, we have explored the influence of this structure on genome translation and replication. We demonstrate that LNAs which block formation of the closed conformation inhibit genome translation. Inhibition was at least partly independent of the initiation mechanism, whether driven by homologous or heterologous internal ribosome entry sites or from a capped message. Provision of SL9266/PK in trans relieved translational inhibition, and mutational analysis implied a mechanism in which the closed conformation recruits a cellular factor that would otherwise suppresses translation. We propose that SL9266/PK functions as a temporal switch, modulating the mutually incompatible processes of translation and replication.
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK a.k.tuplin@leeds.ac.uk.
authors.forename: Andrew
authors.initials: A
authors.lastname: Tuplin
authors.affiliation: School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Struthers
authors.affiliation: School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cook
authors.affiliation: School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Bentley
authors.affiliation: School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK D.J.Evans@Warwick.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Evans
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000235:genetics; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D020413:3' Untranslated Regions; D016376:Oligonucleotides, Antisense; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase