A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors.
Nucleic Acids Res
; 36(15): e95, 2008 Sep.
Article
em En
| MEDLINE
| ID: mdl-18614605
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Biossíntese de Proteínas
/
Regulação Viral da Expressão Gênica
/
Proteínas não Estruturais Virais
/
Hepacivirus
Tipo de estudo:
Clinical_trials
Limite:
Humans
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article