| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Mark Harris
mharris{at}bmb.leeds.ac.uk
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 neoR-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 
-interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture.
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 S. Muller, R. Geffers, and S. Gunther Analysis of gene expression in Lassa virus-infected HuH-7 cells J. Gen. Virol., May 1, 2007; 88(5): 1568 - 1575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. McCormick, S. Maucourant, S. Griffin, D. J. Rowlands, and M. Harris Tagging of NS5A expressed from a functional hepatitis C virus replicon. J. Gen. Virol., March 1, 2006; 87(Pt 3): 635 - 640. [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]
|
|
|
|
|
|
Z. Cai, C. Zhang, K.-S. Chang, J. Jiang, B.-C. Ahn, T. Wakita, T. J. Liang, and G. Luo Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells J. Virol., November 15, 2005; 79(22): 13963 - 13973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang, O. Yamada, T. Sakamoto, H. Yoshida, H. Araki, and K. Shimotohno Exploiting cis-Acting Replication Elements To Direct Hepatitis C Virus-Dependent Transgene Expression J. Virol., May 15, 2005; 79(10): 5923 - 5932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Li, Z. Chen, N. Kato, M. Gale Jr., and S. M. Lemon Distinct Poly(I-C) and Virus-activated Signaling Pathways Leading to Interferon-{beta} Production in Hepatocytes J. Biol. Chem., April 29, 2005; 280(17): 16739 - 16747. [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. Breiman, N. Grandvaux, R. Lin, C. Ottone, S. Akira, M. Yoneyama, T. Fujita, J. Hiscott, and E. F. Meurs Inhibition of RIG-I-Dependent Signaling to the Interferon Pathway during Hepatitis C Virus Expression and Restoration of Signaling by IKK{varepsilon} J. Virol., April 1, 2005; 79(7): 3969 - 3978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Macdonald, S. Mazaleyrat, C. McCormick, A. Street, N. J. Burgoyne, R. M. Jackson, V. Cazeaux, H. Shelton, K. Saksela, and M. Harris Further studies on hepatitis C virus NS5A-SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling J. Gen. Virol., April 1, 2005; 86(4): 1035 - 1044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Macdonald and M. Harris Hepatitis C virus NS5A: tales of a promiscuous protein J. Gen. Virol., September 1, 2004; 85(9): 2485 - 2502. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
