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Journal of Virology, July 2008, p. 7034-7046, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00118-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Efficient trans-Encapsidation of Hepatitis C Virus RNAs into Infectious Virus-Like Particles {triangledown}

Eike Steinmann,1,2,{dagger} Christiane Brohm,1,2,{dagger} Stephanie Kallis,1 Ralf Bartenschlager,1 and Thomas Pietschmann1,2*

Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany,1 Twincore, Center for Experimental and Clinical Infection Research, Department of Experimental Virology, Feodor-Lynen-Strasse 7, 30625 Hannover, Germany2

Received 17 January 2008/ Accepted 1 May 2008

Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCVTCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCVTCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCVTCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 106 tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCVTCP may prove valuable for gene delivery or vaccination approaches.

* Corresponding author. Mailing address: Twincore Center, Department of Experimental Virology, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. Phone: 49 511 220027 130. Fax: 49 511 220027 148. E-mail: pietschmann.thomas{at}mh-hannover.de

{triangledown} Published ahead of print on 14 May 2008.

{dagger} E.S. and C.B. contributed equally to this work.

Journal of Virology, July 2008, p. 7034-7046, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00118-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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