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Journal of Virology, April 2002, p. 4008-4021, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.4008-4021.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Thomas Pietschmann,1 Volker Lohmann,1 Artur Kaul,1 Nicole Krieger,1 Gabriele Rinck,2,{dagger} Gabriel Rutter,3 Dennis Strand,4 and Ralf Bartenschlager1*

Institute for Virology,1 Institute for Internal Medicine, Johannes-Gutenberg University Mainz, 55131 Mainz,4 Department of Molecular Pathology, Heinrich-Pette Institut, 20251 Hamburg, Germany,3 Virco Ltd., Cambridge CB4 0G1, United Kingdom2

Received 31 October 2001/ Accepted 16 January 2002

The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

* Corresponding author. Mailing address: Institute for Virology, Johannes-Gutenberg University Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany. Phone: 49 6131 393 4451. Fax: 49 6131 393 5604. E-mail: bartnsch{at}mail.uni-mainz.de.

{dagger} Present address: Visible Genetics Ltd., 184 Cambridge Science Park, Cambridge CB4 0GA, United Kingdom.

Journal of Virology, April 2002, p. 4008-4021, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.4008-4021.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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Copyright © 2002 by the American Society for Microbiology. All rights reserved.