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Nucleic Acids Research Advance Access published online on July 9, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn427
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© 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.

Methods Online

A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors{dagger}

Loic Jaffrelo1,2, Sandrine Chabas1,2, Sandrine Reigadas1,2, Aude Pflieger1,2, Czeslaw Wychowski3, Julie Rumi4,2, Michel Ventura4,2, Jean-Jacques Toulmé1,2,5 and Cathy Staedel1,2,*

1INSERM U869, F-33076, Bordeaux, France, 2Université Victor Segalen Bordeaux 2, F-33076, Bordeaux, 3UMR-8161 CNRS, Institut de Biologie de Lille-Institut Pasteur de Lille, Lille, 4UMR-5234 CNRS, F-33076, Bordeaux, 5Institut Européen de Chimie et Biologie, 33607 Pessac, France

*To whom correspondence should be addressed. Tel: +33 5 57 57 45 65; +33 5 57 57 10 15; Email: cathy.staedel{at}bordeaux.inserm.fr.

Received February 15, 2008. Revised June 19, 2008. Accepted June 21, 2008.

We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.

{dagger}While this manuscript was being revised, a paper was published describing the anti-HCV properties of a peptide close to W20L (G. Cheng et al., Proc. Natl Acad. Sci., 105, 3088–93). Interestingly, this peptide was identified through a different methodology.


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