Original ResearchBasic and Translational—LiverMouse Hepatic Cells Support Assembly of Infectious Hepatitis C Virus Particles
Section snippets
Cell Culture and Reagents
The cell lines Huh7.5 and Hep56.1D (CLS-Cell Line Service)22 were cultured in Dulbecco's modified minimal essential medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 2 mmol/L L-glutamine, nonessential amino acids, 100 U penicillin per milliliter, 100 μg streptomycin per milliliter, and 10% fetal calf serum (complete DMEM). Huh7.5 and Hep56.1D-derived cell lines containing a subgenomic replicon and the trans-complementing expression cassette were kept in complete DMEM containing 100
Transient and Stable Replication of Genomic HCV RNAs in Mouse Cells
The primary goal of our study was to determine HCV assembly competence of mouse cells for which we selected the cell line Hep56.1D that has excellent growth and transfection properties (not shown). In the initial set of experiments, we transiently transfected Hep56.1D cells with the HCV genome Jc1 and quantified virus production. Although considerable amounts of core protein were produced, no infectivity was detected (data not shown).
Given the very low replication of HCV in mouse cells,20, 21
Discussion
To establish an immunocompetent fully permissive mouse model that is urgently required to study HCV-specific immune responses and pathogenesis, multiple species restrictions need to be overcome. HCV entry into mouse cell lines is possible upon ectopic expression of CD81 and occludin or by using a mouse CD81-adapted HCV genome.16, 17 Whereas HCV internal ribosome entry site (IRES) activity in mouse and human cells appears to be comparable,18, 19 viral RNA replication is very inefficient and
Acknowledgments
The authors thank Ulrike Herian for excellent technical assistance; Susan Uprichard for providing the replicon cell lines MMH1-1 and AML12; Thomas Baumert for provision of SCARB1- and Claudin 1-specific antibodies; Heinrich Wieland for apoE2, 3, 4 constructs; Stephan Urban for providing mouse liver homogenates; Charles M. Rice for Huh7.5 cells and the NS5A-specific antibody 9E10; Takaji Wakita for provision of the original JFH1 clone; and the Nikon Imaging Center at the University of Heidelberg
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M.P.W.'s present address is Applied Molecular Virology, Institute Pasteur Korea, 696 Sampyung-dong Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea.
Conflicts of interest The authors disclose no conflicts.
Funding Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB/TRR77, Teilprojekt 1; to R.B. and V.L.; and TRR83, Teilprojekt 13; to R.B.), by a grant from the European Union (ERC grant contract no. 233130), and by the Marie Curie Training Network EI-HCV, contract no. MRTN-CT-2006-035599 EI-HCV.