title: Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors.
abstract: Baculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a 'minigenome' derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but beta-galactosidase activity was limited to 10-20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis delta virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis delta virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host-cell interactions.
authors.affiliation: Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: McCormick
authors.affiliation: Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rowlands
authors.affiliation: Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1.
authors.forename: Mark
authors.initials: M
authors.lastname: Harris
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D007763:Lac Operon / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D018411:Spodoptera / Q000821:virology; D013752:Tetracycline / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014779:Virus Replication
chemical_list: D004441:Ecdysterone; C008999:ponasterone A; D013752:Tetracycline