HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by McCormick, C. J. Articles by Harris, M. Search for Related Content PubMed PubMed Citation Articles by McCormick, C. J. Articles by Harris, M. Agricola Articles by McCormick, C. J. Articles by Harris, M.

Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors

Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK¹

Author for correspondence: Mark Harris. Fax +44 113 233 5638. e-mail mharris{at}bmb.leeds.ac.uk

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 -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 virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis 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.

This article has been cited by other articles:

				R. Adair, A. H. Patel, L. Corless, S. Griffin, D. J. Rowlands, and C. J. McCormick Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA J. Gen. Virol., April 1, 2009; 90(4): 833 - 842. [Abstract] [Full Text] [PDF]

				C. J. McCormick, O. Salim, P. R. Lambden, and I. N. Clarke Translation Termination Reinitiation between Open Reading Frame 1 (ORF1) and ORF2 Enables Capsid Expression in a Bovine Norovirus without the Need for Production of Viral Subgenomic RNA J. Virol., September 1, 2008; 82(17): 8917 - 8921. [Abstract] [Full Text] [PDF]

				M. Kitajima and H. Takaku Induction of Antitumor Acquired Immunity by Baculovirus Autographa californica Multiple Nuclear Polyhedrosis Virus Infection in Mice Clin. Vaccine Immunol., February 1, 2008; 15(2): 376 - 378. [Abstract] [Full Text] [PDF]

				V. K. Ward, C. J. McCormick, I. N. Clarke, O. Salim, C. E. Wobus, L. B. Thackray, H. W. Virgin IV, and P. R. Lambden Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA PNAS, June 26, 2007; 104(26): 11050 - 11055. [Abstract] [Full Text] [PDF]

				C. J. McCormick, D. Brown, S. Griffin, L. Challinor, D. J. Rowlands, and M. Harris A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A J. Gen. Virol., January 1, 2006; 87(1): 93 - 102. [Abstract] [Full Text] [PDF]

				A. Street, A. Macdonald, C. McCormick, and M. Harris Hepatitis C Virus NS5A-Mediated Activation of Phosphoinositide 3-Kinase Results in Stabilization of Cellular {beta}-Catenin and Stimulation of {beta}-Catenin-Responsive Transcription J. Virol., April 15, 2005; 79(8): 5006 - 5016. [Abstract] [Full Text] [PDF]

				A. Street, A. Macdonald, K. Crowder, and M. Harris The Hepatitis C Virus NS5A Protein Activates a Phosphoinositide 3-Kinase-dependent Survival Signaling Cascade J. Biol. Chem., March 26, 2004; 279(13): 12232 - 12241. [Abstract] [Full Text] [PDF]

				C. J. McCormick, L. Challinor, A. Macdonald, D. J. Rowlands, and M. Harris Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system J. Gen. Virol., February 1, 2004; 85(2): 429 - 439. [Abstract] [Full Text] [PDF]

				A. Macdonald, K. Crowder, A. Street, C. McCormick, K. Saksela, and M. Harris The Hepatitis C Virus Non-structural NS5A Protein Inhibits Activating Protein-1 Function by Perturbing Ras-ERK Pathway Signaling J. Biol. Chem., May 9, 2003; 278(20): 17775 - 17784. [Abstract] [Full Text] [PDF]