A HUMAN LIVER-CELL LINE EXHIBITS EFFICIENT TRANSLATION OF HCV RNAS PRODUCED BY A RECOMBINANT ADENOVIRUS EXPRESSING T7 RNA-POLYMERASE

An in vitro system that supports the efficient growth of hepatitis C v irus (HCV) and reflects its complete in vitro replication cycle has no t 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 replica tion-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 inocula tion. 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'UT R of HCV, a ribozyme of hepatitis D virus and a T7 terminator (pT7HCVL uc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with pT7HCVLuc. Howeve r, 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 synthesized in vitro from PT7HCVLuc i nduced a higher level of reporter gene expression than RNA from pT7EMC VLuc. The T7-adenovirus system for the synthesis of HCV minigenes in v ivo provides useful information on the molecular mechanisms of HCV tra nslation in human liver cells. (C0 1998 Academic Press.