A Human Liver Cell Line Exhibits Efficient Translation of HCV RNAs Produced by a Recombinant Adenovirus Expressing T7 RNA Polymerase

Abstract

Anin vitrosystem that supports the efficient growth of hepatitis C virus (HCV) and reflects its completein vitroreplication cycle has not yet been established. The establishment of a minigene RNA of HCV in mammalian cells could facilitate the study of virus–cell interactions and the molecular pathogenesis of this virus. We constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). A high level of T7 RNA polymerase was detectable for at least 11 days after inoculation. Cells infected with AdexCAT7 were then transfected with plasmids carrying the authentic T7 promoter, the 5′ untranslated region (UTR) of encephalomyocarditis virus, a luciferase gene, and a T7 terminator (pT7EMCVLuc) or carrying the modified T7 promoter, the 5′UTR of HCV, a luciferase gene, the coding region of C-terminal of NS5B and the 3′UTR of HCV, a ribozyme of hepatitis D virus and a T7 terminator (pT7HCVLuc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with pT7HCVLuc. However, one cell line, FLC4, derived from a human hepatocellular carcinoma, exhibited very high reporter gene expression with pT7HCVLuc. In this cell line, transfection with RNA synthesizedin vitrofrom pT7HCVLuc induced a higher level of reporter gene expression than RNA from pT7EMCVLuc. The T7-adenovirus system for the synthesis of HCV minigenesin vivoprovides useful information on the molecular mechanisms of HCV translation in human liver cells.