Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP |
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Authors: | Masashi Iwamoto Koichi Watashi Senko Tsukuda Hussein Hassan Aly Masayoshi Fukasawa Akira Fujimoto Ryosuke Suzuki Hideki Aizaki Takayoshi Ito Osamu Koiwai Hiroyuki Kusuhara Takaji Wakita |
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Institution: | 1. Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan;2. Department of Applied Biological Science, Tokyo University of Sciences, Noda 278-8510, Japan;3. Micro-signaling Regulation Technology Unit, RIKEN Center for Life Science Technologies, Wako 351-0198, Japan;4. Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan;5. Division of Gastroenterology, Department of Medicine, Showa University School of Medicine, Tokyo 142-8666, Japan;6. The University of Tokyo, Graduate School of Pharmaceutical Sciences, Tokyo 113-0033, Japan |
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Abstract: | Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection. |
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Keywords: | Ab antibody cccDNA covalently closed circular DNA Cs cyclosporin DMSO dimethyl sulfoxide GEq genome equivalent HBc HBV core protein HBs HBV surface protein HBV hepatitis B virus NTCP sodium taurocholate cotransporting polypeptide OHC hydroxycholesterol PHH primary human hepatocytes PTH primary tupaia hepatocytes |
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