Nucleic Acids Research Advance Access originally published online on December 23, 2008
Nucleic Acids Research 2009 37(3):957-971; doi:10.1093/nar/gkn1022
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Nucleic Acids Research, 2009, Vol. 37, No. 3 957-971
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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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
1Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Facultad de Medicina, Pontificia Universidad Católica de Chile, 2Jeunes Equipes Associées à l’IRD y Nucleo Milenio de Inmunología e Inmunoterapia, 3Centre for Bioinformatics (CBUC), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 4Departamento de Biología, Facultad de Ciencias, Universidad de Chile, 5Fundación Ciencia para la Vida and 6Departamento de Gastroenterología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
*To whom correspondence should be addressed. Tel: +56 2 354 3410; Fax: +56 2 638 7457; Email: malopez{at}med.puc.cl
Received November 5, 2008. Revised December 7, 2008. Accepted December 8, 2008.
The HCV internal ribosome entry site (IRES) spans a region of 
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 Mg2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.
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