Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells
Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG), hNTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line (Huh7DhNTCP) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO / 4% PEG8000, and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7DhNTCP) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7DhNTCP cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7DhNTCP cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO / 4% PEG8000, whereas the others were not. Huh7DhNTCP cells were established to express a high level of hNTCP compared to liver extracts, and Huh7DhNTCP cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7DhNTCP cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.
KeywordsHepatitis B virus (HBV) Na+/taurocholate cotransporting polypeptide (NTCP) Huh7 dimethyl sulfoxide (DMSO) polyethylene glycol (PEG) susceptibility
- Cao L, Wu C, Shi H, Gong Z, Zhang E, Wang H, Zhao K, Liu S, Li S, Gao X, Wang Y, Pei R, Lu M, Chen X. 2014. Coexistence of hepatitis B virus quasispecies enhances viral replication and the ability to induce host antibody and cellular immune responses. J Virol, 88: 8656–8666.CrossRefPubMedPubMedCentralGoogle Scholar
- Lempp FA, Wiedtke E, Qu B, Roques P, Chemin I, Vondran FW, Le Grand R, Grimm D, Urban S. 2017. Sodium taurocholate cotransporting polypeptide is the limiting host factor of Hepatitis B Virus infection in macaque and pig hepatocytes. Hepatology. doi: 10.1002/hep.29112.Google Scholar
- Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M, Stindt J, Koniger C, Nassal M, Kubitz R, Sultmann H, Urban S. 2014. Hepatitis B and D viruses exploit sodium taurocholate cotransporting polypeptide for species-specific entry into hepatocytes. Gastroenterology, 146: 1070–1083.CrossRefPubMedGoogle Scholar
- Schulze-Bergkamen H, Untergasser A, Dax A, Vogel H, Buchler P, Klar E, Lehnert T, Friess H, Buchler MW, Kirschfink M, Stremmel W, Krammer PH, Muller M, Protzer U. 2003. Primary human hepatocytes—a valuable tool for investigation of apoptosis and hepatitis B virus infection. J Hepatol, 38: 736–744.CrossRefPubMedGoogle Scholar
- Shimura S, Watashi K, Fukano K, Peel M, Sluder A, Kawai F, Iwamoto M, Tsukuda S, Takeuchi JS, Miyake T, Sugiyama M, Ogasawara Y, Park SY, Tanaka Y, Kusuhara H, Mizokami M, Sureau C, Wakita T. 2017. Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity. J Hepatol, 66: 685–692.CrossRefPubMedGoogle Scholar
- Tian Y, James Ou J. Genetic and epigenetic alterations in hepatitis B virus-associated hepatocellular carcinoma. Virol Sin(30): 85–91.Google Scholar
- Wu C, Zhang X, Tian Y, Song J, Yang D, Roggendorf M, Lu M, Chen X. 2010. Biological significance of amino acid substitutions in hepatitis B surface antigen (HBsAg) for glycosylation, secretion, antigenicity and immunogenicity of HBsAg and hepatitis B virus replication. J Gen Virol, 91: 483–492.CrossRefPubMedGoogle Scholar
- Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W. 2012. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife, 1: e00049.CrossRefPubMedPubMedCentralGoogle Scholar
OPEN ACCESS This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.