Abstract
Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for hepatitis B virus (HBV). Expressing human NTCP in human hepatoma HepG2 cells (HepG2-NTCP) renders these cells susceptible for HBV infection. The HepG2-NTCP stably transfected cell line provides a much-needed and easily accessible platform for studying the virus. HepG2-NTCP cells could also be used to identify chemicals targeting key steps of the virus life cycle including HBV covalent closed circular (ccc) DNA, and enable the development of novel antivirals against the infection.
Many factors may contribute to the efficiency of HBV infection on HepG2-NTCP cells, with clonal differences among cell line isolates, the source of viral inoculum, and infection medium among the most critical ones. Here, we provide detailed protocols for efficient HBV infection of HepG2-NTCP cells in culture; generation and selection of single cell clones of HepG2-NTCP; production of infectious HBV virion stock through DNA transfection of recombinant plasmid that enables studying primary clinical HBV isolates; and assessing the infection with immunostaining of HBV antigens and Southern blot analysis of HBV cccDNA.
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Acknowledgment
The work was supported by the National Science and Technology Major Project, China (2013ZX09509102), the Ministry of Science and Technology, China (2014CB 849601), and the Science and Technology Bureau of Beijing Municipal Government. We thank Ju-Tao Guo for helpful discussions on Southern blot analysis of cccDNA. We thank Xiaofeng Feng for cloning the 1.05 copies of HBV genome (genotype D), Jianhua Sui and Zhiliang Cao for mcAbs production, Guocai Zhong, Huan Yan, and Zhenchao Gao for setting up or optimizing the infection procedure.
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Sun, Y., Qi, Y., Peng, B., Li, W. (2017). NTCP-Reconstituted In Vitro HBV Infection System. In: Guo, H., Cuconati, A. (eds) Hepatitis B Virus. Methods in Molecular Biology, vol 1540. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6700-1_1
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DOI: https://doi.org/10.1007/978-1-4939-6700-1_1
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