title: Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation.
abstract: The HCV internal ribosome entry site (IRES) spans a region of approximately 340 nt that encompasses most of the 5' untranslated region (5'UTR) of the viral mRNA and the first 24-40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5'UTR on IRES activity, naturally occurring variants of the 5'UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg(2+) ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.
authors.affiliation: Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors.forename: María Inés
authors.initials: MI
authors.lastname: Barría
authors.forename: Angel
authors.initials: A
authors.lastname: González
authors.forename: Jorge
authors.initials: J
authors.lastname: Vera-Otarola
authors.forename: Ursula
authors.initials: U
authors.lastname: León
authors.forename: Valeska
authors.initials: V
authors.lastname: Vollrath
authors.forename: Delphine
authors.initials: D
authors.lastname: Marsac
authors.forename: Octavio
authors.initials: O
authors.lastname: Monasterio
authors.forename: Tomás
authors.initials: T
authors.lastname: Pérez-Acle
authors.forename: Alejandro
authors.initials: A
authors.lastname: Soza
authors.forename: Marcelo
authors.initials: M
authors.lastname: López-Lastra
mesh_terms: D020121:5' Untranslated Regions; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D016174:Hepacivirus / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000821:virology; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D012315:RNA Caps / Q000378:metabolism; D012367:RNA, Viral / Q000097:blood / Q000737:chemistry*; D038621:Regulatory Sequences, Ribonucleic Acid
chemical_list: D020121:5' Untranslated Regions; D012315:RNA Caps; D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid