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Nucleic Acids Research Advance Access published online on November 4, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp890
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

Maricarmen Vallejos1, Pablo Ramdohr1, Fernando Valiente-Echeverría1, Karla Tapia1, Felipe E. Rodriguez2, Fernando Lowy1, J. Pablo Huidobro-Toro2, John A. Dangerfield3 and Marcelo López-Lastra1,*

1Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Pontificia Universidad Católica de Chile, Marcoleta 391, 2Centro de Regulación Celular y Patología, J. V. Luco e Instituto Milenio de Biología Fundamental y Aplicada, MIFAB, Departamento de Fisiología, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile and 3Institute of Virology, University of Veterinary Sciences, Veterinaerplatz 1, A-1210 Vienna, Austria and Christian Doppler Laboratory Foreign Module for Virology-Nanotechnology, #05-518 Centros, 20 Biopolis Way, 138668 Singapore

*To whom correspondence should be addressed. Tel: +56 2 3543410; Fax: +56 2 638 7457; Email: malopez{at}med.puc.cl

Received May 7, 2009. Revised September 19, 2009. Accepted October 5, 2009.

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m7GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.


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