title: Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system.
abstract: We have developed a baculovirus delivery system that enables tetracycline-regulated expression of polII-derived hepatitis C virus (HCV) transcripts in hepatocyte-derived cell lines (McCormick et al., 2002). As part of a study to determine whether such transcripts are replication competent, the transcription start site of the tetracycline-regulable promoter was mapped and three baculovirus transfer vectors containing a neo(R)-expressing culture adapted replicon cDNA were generated. These vectors either had the first nucleotide of the 5'UTR positioned -2 (mkI) and +1 (mkII) with respect to the transcription start site, or included a hammerhead ribozyme at the 5' end of the transcript (5'HH) that cleaves between the ribozyme-5'UTR boundary. Transfection of all of the culture-adapted replicon constructs into Huh7 cells resulted in the formation of more neomycin-resistant colonies than seen with a polymerase knock-out replicon construct, although this was less pronounced in the mkI group. Furthermore, both the positive- and negative-strands of the replicon could be detected in all neomycin-resistant polyclonal cell lines except for those derived from transfection of the polymerase knock-out construct. Transduction of Huh7 cells with recombinant baculoviruses carrying the same expression cassettes improved replicon delivery, but the relative efficiency of the constructs remained the same. The baculovirus vectors were also used to introduce the replicon transcript into HepG2 cells. Expression of the culture-adapted but not the polymerase knock-out construct induced transcription of the beta-interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture.
authors.affiliation: Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
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, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Challinor
authors.affiliation: Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Macdonald
authors.affiliation: Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
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, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Harris
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016865:Reassortant Viruses / Q000235:genetics*; D012093:Replicon; D013752:Tetracycline; D014161:Transduction, Genetic; D014779:Virus Replication
chemical_list: D020121:5' Untranslated Regions; D013752:Tetracycline