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Journal of Virology, June 2006, p. 5308-5320, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02460-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of the Early Steps of Hepatitis C Virus Infection by Using Luciferase Reporter Viruses

George Koutsoudakis, Artur Kaul, Eike Steinmann, Stephanie Kallis, Volker Lohmann, Thomas Pietschmann,* and Ralf Bartenschlager*

Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany

Received 22 November 2005/ Accepted 18 February 2006

The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors.

* Corresponding author. Mailing address: Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, Heidelberg 69120, Germany. Fax: 49 6221 564570. Phone for T. Pietschmann: 49 6221 566449. E-mail for T. Pietschmann: thomas_pietschmann{at}med.uni-heidelberg.de. Phone for R. Bartenschlager: 49 6221 564569. E-mail for R. Bartenschlager: ralf_bartenschlager{at}med.uni-heidelberg.de.

Journal of Virology, June 2006, p. 5308-5320, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02460-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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