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REPLICATION

Persistent Replication of Hepatitis C Virus Replicons Expressing the β-Lactamase Reporter in Subpopulations of Highly Permissive Huh7 Cells

Edward M. Murray, Jay A. Grobler, Eric J. Markel, Marco F. Pagnoni, Giacomo Paonessa, Adam J. Simon, Osvaldo A. Flores
Edward M. Murray
1Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486
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Jay A. Grobler
1Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486
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Eric J. Markel
1Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486
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Marco F. Pagnoni
1Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486
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Giacomo Paonessa
2Istituto di Ricerche di Biologia Molecolare P. Angeletti, 00040 Pomezia (Rome), Italy
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Adam J. Simon
1Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486
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Osvaldo A. Flores
1Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486
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  • For correspondence: osvaldo_flores@merck.com
DOI: 10.1128/JVI.77.5.2928-2935.2003
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ABSTRACT

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the β-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or “enhanced”) to HCV replication than naïve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among naïve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the β-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.

  • Copyright © 2003 American Society for Microbiology
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Persistent Replication of Hepatitis C Virus Replicons Expressing the β-Lactamase Reporter in Subpopulations of Highly Permissive Huh7 Cells
Edward M. Murray, Jay A. Grobler, Eric J. Markel, Marco F. Pagnoni, Giacomo Paonessa, Adam J. Simon, Osvaldo A. Flores
Journal of Virology Mar 2003, 77 (5) 2928-2935; DOI: 10.1128/JVI.77.5.2928-2935.2003

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Persistent Replication of Hepatitis C Virus Replicons Expressing the β-Lactamase Reporter in Subpopulations of Highly Permissive Huh7 Cells
Edward M. Murray, Jay A. Grobler, Eric J. Markel, Marco F. Pagnoni, Giacomo Paonessa, Adam J. Simon, Osvaldo A. Flores
Journal of Virology Mar 2003, 77 (5) 2928-2935; DOI: 10.1128/JVI.77.5.2928-2935.2003
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KEYWORDS

Genes, Reporter
hepacivirus
replicon
virus replication
beta-Lactamases

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