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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.

We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells. Two recombinant baculoviruses carrying cDNA of bacteriophage T7 RNA polymerase, with or without a nuclear localization signal, under the control of a mammalian promoter were constructed. High level expression of T7 RNA polymerase was observed in various mammalian cell lines after infection with the recombinant baculoviruses. After transfection of plasmids containing the luciferase gene under the control of the T7 promoter, high luciferase activity was detected in cells infected with the recombinant baculoviruses. We also constructed a plasmid containing an entire cDNA clone of type 1 poliovirus under the T7 promoter. Two days after transfection of the plasmid into the cells infected with the recombinant baculoviruses, a high titer of poliovirus was recovered. The use of the recombinant baculoviruses did not cause any cytopathic effects even at a high multiplicity of infection. The lack of replication ability and low toxicity are the advantageous features of the hybrid baculovirus–T7 polymerase system in comparison with the widely used vaccinia–T7 polymerase system for gene expression and recovery of infectious viruses from its cDNA.