A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors |
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Authors: | Jaffrelo Loic Chabas Sandrine Reigadas Sandrine Pflieger Aude Wychowski Czeslaw Rumi Julie Ventura Michel Toulmé Jean-Jacques Staedel Cathy |
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Affiliation: | Loic Jaffrelo, Sandrine Chabas, Sandrine Reigadas, Aude Pflieger, Czeslaw Wychowski, Julie Rumi, Michel Ventura, Jean-Jacques Toulmé, and Cathy Staedel |
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Abstract: | 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. |
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