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Journal of Virology, March 2003, p. 3007-3019, Vol. 77, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.5.3007-3019.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture

Volker Lohmann,1 Sandra Hoffmann,2 Ulrike Herian,1 Francois Penin,3 and Ralf Bartenschlager1*

Department of Molecular Virology, Otto-Meyerhof-Zentrum, Im Neuenheimer Feld 350, 69120 Heidelberg,1 Institute for Virology, Johannes-Gutenberg University Mainz, 55131 Mainz, Germany,2 Institut de Biologie et Chimie des Proteines, CNRS UMR5086, 69367 Lyon Cedex 07, France3

Received 20 September 2002/ Accepted 21 November 2002

Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.

* Corresponding author. Mailing address: Department of Molecular Virology, Otto-Meyerhof-Zentrum, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany. Phone: 49-6221-564569. Fax: 49-6221-564570. E-mail: ralf_bartenschlager{at}med.uni-heidelberg.de.

Journal of Virology, March 2003, p. 3007-3019, Vol. 77, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.5.3007-3019.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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