Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

• Published in: Scientific reports Vol. 6; no. 1; p. 22952
• Main Authors: Stewart, H., Bingham, R.J., White, S. J., Dykeman, E. C., Zothner, C., Tuplin, A. K., Stockley, P. G., Twarock, R., Harris, M.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14-03-2016; Nature Publishing Group
• Subjects: 45/91; 631/326/421; 631/326/596/1905; 82/80; Aptamers; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - genetics; Aptamers, Nucleotide - metabolism; Base Sequence; Blotting, Western; Cell Line, Tumor; Core protein; Encapsidation; Gene Expression Regulation, Viral; Genome, Viral - genetics; Genomes; Hepacivirus - genetics; Hepacivirus - metabolism; Hepatitis; Hepatitis C; Humanities and Social Sciences; Humans; Infectivity; multidisciplinary; Mutation; Nucleic Acid Conformation; Nucleotide Motifs - genetics; Packaging; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA, Viral - chemistry; RNA, Viral - genetics; RNA, Viral - metabolism; Science; SELEX Aptamer Technique; Statistical analysis; Viral Core Proteins - genetics; Viral Core Proteins - metabolism; Viral Proteins - genetics; Viral Proteins - metabolism; Virions; Virus Assembly - genetics; Viruses
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep22952

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.