title: A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors.
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.
authors.affiliation: INSERM U869, F-33076, Bordeaux, France.
authors.forename: Loic
authors.initials: L
authors.lastname: Jaffrelo
authors.forename: Sandrine
authors.initials: S
authors.lastname: Chabas
authors.forename: Sandrine
authors.initials: S
authors.lastname: Reigadas
authors.forename: Aude
authors.initials: A
authors.lastname: Pflieger
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Wychowski
authors.forename: Julie
authors.initials: J
authors.lastname: Rumi
authors.forename: Michel
authors.initials: M
authors.lastname: Ventura
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Toulmé
authors.forename: Cathy
authors.initials: C
authors.lastname: Staedel
mesh_terms: D020121:5' Untranslated Regions / Q000737:chemistry; D002460:Cell Line; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral; D015723:Gene Library; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry; D038621:Regulatory Sequences, Ribonucleic Acid; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase