Gastroenterology

Gastroenterology

Volume 141, Issue 3, September 2011, Pages 1057-1066
Gastroenterology

Original Research
Basic and Translational—Liver
Mouse Hepatic Cells Support Assembly of Infectious Hepatitis C Virus Particles

https://doi.org/10.1053/j.gastro.2011.06.010Get rights and content

Background & Aims

Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines.

Methods

A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (apoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant.

Results

Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. Thus, apoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of apoE and mouse apoE support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE.

Conclusions

Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection.

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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  • Cited by (0)

    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.

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