Identification of eIF2B gamma and eIF2 gamma as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

The 5'-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribo some entry site (IRES) to initiate translation of HCV proteins. We have dev eloped a selection system based on a randomized hairpin ribozyme gene libra ry to identify cellular factors involved in HCV IREs function. A retroviral vector ribozyme library with randomized target recognition sequences was i ntroduced into HeLa cells, stably expressing a bicistronic construct encodi ng the hygromycin B phosphotransferase gene and the herpes simplex virus th ymidine kinase gene (HSV-tk). Translation of the HSV-fk gene was mediated b y the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated t ranslation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-res istant phenotype and were shown to inhibit IREs-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the g amma subunits of human eukaryotic initiation factors 2B (elF2B gamma) and 2 (elF2 gamma), respectively. The involvement of elF2B gamma and elF2 gamma in HCV IREs-mediated translation was further validated by ribozymes directe d against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IREs cofactors, ribozymes obtaine d from this cellular selection system could be directly used to specificall y inhibit HCV viral translation, thereby facilitating the development of ne w antiviral strategies for HCV infection.