title: Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging.
abstract: The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly.
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: H
authors.initials: H
authors.lastname: Stewart
authors.affiliation: York Centre for Complex Systems Analysis, Departments of Mathematics and Biology, University of York, York, YO10 5DD, United Kingdom.
authors.forename: R J
authors.initials: RJ
authors.lastname: Bingham
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: S J
authors.initials: SJ
authors.lastname: White
authors.affiliation: York Centre for Complex Systems Analysis, Departments of Mathematics and Biology, University of York, York, YO10 5DD, United Kingdom.
authors.forename: E C
authors.initials: EC
authors.lastname: Dykeman
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: C
authors.initials: C
authors.lastname: Zothner
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.|Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: A K
authors.initials: AK
authors.lastname: Tuplin
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: P G
authors.initials: PG
authors.lastname: Stockley
authors.affiliation: York Centre for Complex Systems Analysis, Departments of Mathematics and Biology, University of York, York, YO10 5DD, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Twarock
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.|Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Harris
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D052156:SELEX Aptamer Technique; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly / Q000235:genetics*
chemical_list: D052157:Aptamers, Nucleotide; D012367:RNA, Viral; D014758:Viral Core Proteins; D014764:Viral Proteins