For best viewing of the website please use Mozilla Firefox or Google Chrome.
Citation: Yingying Song, Shuyu Shou, Huimin Guo, Zixiang Gao, Nannan Liu, Yang Yang, Feifei Wang, Qiang Deng, Jing Liu, Youhua Xie. Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection [J].VIROLOGICA SINICA, 2022, 37(4) : 558-568.  http://dx.doi.org/10.1016/j.virs.2022.05.002

Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection

  • Hepatitis B virus (HBV) is a primary cause of chronic liver diseases in humans. HBV infection exhibits strict host and tissue tropism. HBV core promoter (Cp) drives transcription of pregenomic RNA (pgRNA) and plays a key role in the viral life cycle. Hepatocyte nuclear factor 4α (HNF4α) acts as a major transcriptional factor that stimulates Cp. In this work, we reported that BEL7404 cell line displayed a high efficiency of DNA transfection and high levels of HBV antigen expression after transfection of HBV replicons without prominent viral replication. The introduction of exogenous HNF4α and human sodium taurocholate cotransporting polypeptide (hNTCP) expression into BEL7404 made it permissive for HBV replication and susceptible to HBV infection. BEL7404-derived cell lines with induced HBV permissiveness and susceptibility were constructed by stable co-transfection of hNTCP and Tet-inducible HNF4α followed by limiting dilution cloning. HBV replication in such cells was sensitive to inhibition by nucleotide analog tenofovir, while the infection was inhibited by HBV entry inhibitors. This cell culture system provides a new and additional tool for the study of HBV replication and infection as well as the characterization of antiviral agents.

  • 加载中
  • 10.1016j.virs.2022.05.002-ESM.docx
    1. Bai, W., Cui, X., Chen, R., Tao, S., Hong, R., Zhang, J., Zhang, J., Wang, Y., Xie, Y., Liu, J., 2016. Re-designed recombinant hepatitis b virus vectors enable efficient delivery of versatile cargo genes to hepatocytes with improved safety. Viruses 8, 129.

    2. Chang, H.K., Wang, B.Y., Yuh, C.H., Wei, C.L., Ting, L.P., 1989. A liver-specific nuclear factor interacts with the promoter region of the large surface protein gene of human hepatitis b virus. Mol. Cell Biol. 9, 5189–5197.

    3. Chen, R., Zhu, D., Ye, X., Shen, D., Lu, R., 1980. Establishment of three human liver carcinoma cell lines and some of their biological characteristics in vitro. Sci. Sin. 23, 236–247.

    4. Gao, W., Hu, J., 2007. Formation of hepatitis b virus covalently closed circular DNA:removal of genome-linked protein. J. Virol. 81, 6164–6174.

    5. Garcia, A.D., Ostapchuk, P., Hearing, P., 1993. Functional interaction of nuclear factors ef-c, hnf-4, and rxr alpha with hepatitis b virus enhancer i. J. Virol. 67, 3940–3950.

    6. Gripon, P., Rumin, S., Urban, S., Le Seyec, J., Glaise, D., Cannie, I., Guyomard, C., Lucas, J., Trepo, C., Guguen-Guillouzo, C., 2002. Infection of a human hepatoma cell line by hepatitis b virus. Proc. Natl. Acad. Sci. U. S. A. 99, 15655–15660.

    7. Guo, H., Mao, R., Block, T.M., Guo, J.T., 2010. Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. J. Virol. 84, 387–396.

    8. Guo, H., Jiang, D., Zhou, T., Cuconati, A., Block, T.M., Guo, J.T., 2007. Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis b virus: an intermediate of covalently closed circular DNA formation. J. Virol. 81, 12472–12484.

    9. Hirt, B., 1967. Selective extraction of polyoma DNA from infected mouse cell cultures.J. Mol. Biol. 26, 365–369.

    10. Huang, Y., Liu, Y., Zheng, C., Shen, C., 2017. Investigation of cross-contamination and misidentification of 278 widely used tumor cell lines. PLoS One 12, e0170384.

    11. Iwamoto, M., Watashi, K., Tsukuda, S., Aly, H.H., Fukasawa, M., Fujimoto, A., Suzuki, R., Aizaki, H., Ito, T., Koiwai, O., Kusuhara, H., Wakita, T., 2014. Evaluation and identification of hepatitis b virus entry inhibitors using hepg2 cells overexpressing a membrane transporter ntcp. Biochem. Biophys. Res. Commun. 443, 808–813.

    12. Jia, L., Gao, Y., He, Y., Hooper, J.D., Yang, P., 2020. Hbv induced hepatocellular carcinoma and related potential immunotherapy. Pharmacol. Res. 159, 104992.

    13. Konig, A., Doring, B., Mohr, C., Geipel, A., Geyer, J., Glebe, D., 2014. Kinetics of the bile acid transporter and hepatitis b virus receptor naþ/taurocholate cotransporting polypeptide (ntcp) in hepatocytes. J. Hepatol. 61, 867–875.

    14. Konig, A., Yang, J., Jo, E., Park, K.H.P., Kim, H., Than, T.T., Song, X.Y., Qi, X.X., Dai, X.H., Park, S., Shum, D., Ryu, W.S., Kim, J.H., Yoon, S.K., Park, J.Y., Ahn, S.H., Han, K.H., Gerlich, W.H., Windisch, M.P., 2019. Efficient long-term amplification of hepatitis b virus isolates after infection of slow proliferating hepg2-ntcp cells. J. Hepatol. 71, 289–300.

    15. Ladner, S.K., Otto, M.J., Barker, C.S., Zaifert, K., Wang, G.H., Guo, J.T., Seeger, C., King, R.W., 1997. Inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication. Antimicrob. Agents Chemother. 41, 1715–1720.

    16. Lin, J., Gu, C., Shen, Z., Liu, Y., Wang, W., Tao, S., Cui, X., Liu, J., Xie, Y., 2017.Hepatocyte nuclear factor 1alpha downregulates hbv gene expression and replication by activating the nf-kappab signaling pathway. PLoS One 12, e0174017.

    17. Lopez-Cabrera, M., Letovsky, J., Hu, K.Q., Siddiqui, A., 1990. Multiple liver-specific factors bind to the hepatitis b virus core/pregenomic promoter: trans-activation and repression by ccaat/enhancer binding protein. Proc. Natl. Acad. Sci. U. S. A. 87, 5069–5073.

    18. Lutgehetmann, M., Mancke, L.V., Volz, T., Helbig, M., Allweiss, L., Bornscheuer, T., Pollok, J.M., Lohse, A.W., Petersen, J., Urban, S., Dandri, M., 2012. Humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation. Hepatology 55, 685–694.

    19. Nassal, M., Rieger, A., 1996. A bulged region of the hepatitis b virus rna encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis.J. Virol. 70, 2764–2773.

    20. Polaris Observatory, C., 2018. Global prevalence, treatment, and prevention of hepatitis b virus infection in 2016: a modelling study. Lancet Gastroenterol. Hepatol. 3, 383–403.

    21. Qiao, L., Sui, J., Luo, G., 2018. Robust human and murine hepatocyte culture models of hepatitis b virus infection and replication. J. Virol. 92 e01255-18.

    22. Qin, J., Zhai, J., Hong, R., Shan, S., Kong, Y., Wen, Y., Wang, Y., Liu, J., Xie, Y., 2009.Prospero-related homeobox protein (prox1) inhibits hepatitis b virus replication through repressing multiple cis regulatory elements. J. Gen. Virol. 90, 1246–1255.

    23. Raney, A.K., Easton, A.J., Milich, D.R., McLachlan, A., 1991. Promoter-specific transactivation of hepatitis b virus transcription by a glutamine- and proline-rich domain of hepatocyte nuclear factor 1. J. Virol. 65, 5774–5781.

    24. Raney, A.K., Kline, E.F., Tang, H., McLachlan, A., 2001. Transcription and replication of a natural hepatitis b virus nucleocapsid promoter variant is regulated in vivo by peroxisome proliferators. Virology 289, 239–251.

    25. Rebouissou, S., Zucman-Rossi, J., Moreau, R., Qiu, Z.X., Hui, L.J., 2017. Note of caution:contaminations of hepatocellular cell lines. J. Hepatol. 67, 896–897.

    26. Schrem, H., Klempnauer, J., Borlak, J., 2002. Liver-enriched transcription factors in liver function and development. Part i: the hepatocyte nuclear factor network and liverspecific gene expression. Pharmacol. Rev. 54, 129–158.

    27. Schrem, H., Klempnauer, J., Borlak, J., 2004. Liver-enriched transcription factors in liver function and development. Part ii: the c/ebps and d site-binding protein in cell cycle control, carcinogenesis, circadian gene regulation, liver regeneration, apoptosis, and liver-specific gene regulation. Pharmacol. Rev. 56, 291–330.

    28. Schulze, A., Schieck, A., Ni, Y., Mier, W.,, Urban, S., 2010. Fine mapping of pre-s sequence requirements for hepatitis b virus large envelope protein-mediated receptor interaction. J. Virol. 84, 1989–2000.

    29. Seeger, C., Mason, W.S., 2000. Hepatitis b virus biology. Microbiol. Mol. Biol. Rev. 64, 51–68.

    30. Shen, Z., Yang, H., Yang, S., Wang, W., Cui, X., Zhou, X., Liu, W., Pan, S., Liu, Y., Zhang, J., Zhang, J., Xie, Y., Liu, J., 2017. Hepatitis b virus persistence in mice reveals il-21 and il-33 as regulators of viral clearance. Nat. Commun. 8, 2119.

    31. Tan, M., Bhadoria, A.S., Cui, F., Tan, A., Van Holten, J., Easterbrook, P., Ford, N., Han, Q., Lu, Y., Bulterys, M., Hutin, Y., 2021. Estimating the proportion of people with chronic hepatitis b virus infection eligible for hepatitis b antiviral treatment worldwide: a systematic review and meta-analysis. Lancet Gastroenterol. Hepatol. 6, 106–119.

    32. Tang, H., McLachlan, A., 2001. Transcriptional regulation of hepatitis b virus by nuclear hormone receptors is a critical determinant of viral tropism. Proc. Natl. Acad. Sci. U.S. A. 98, 1841–1846.

    33. Tang, H., McLachlan, A., 2002. Mechanisms of inhibition of nuclear hormone receptordependent hepatitis b virus replication by hepatocyte nuclear factor 3beta. J. Virol. 76, 8572–8581.

    34. Tang, H., Banks, K.E., Anderson, A.L., McLachlan, A., 2001. Hepatitis b virus transcription and replication. Drug News Perspect. 14, 325–334.

    35. Trepo, C., Chan, H.L., Lok, A., 2014. Hepatitis b virus infection. Lancet 384, 2053–2063.

    36. Wang, W., Sun, L., Li, T., Ma, Y., Li, J., Liu, Y., Li, M., Wang, L., Li, C., Xie, Y., Wen, Y., Liang, M., Chen, L., Tong, S., 2016. A human monoclonal antibody against small envelope protein of hepatitis b virus with potent neutralization effect. mAbs 8, 468–477.

    37. 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.

    38. Yu, X., Mertz, J.E., 1996. Promoters for synthesis of the pre-c and pregenomic mrnas of human hepatitis b virus are genetically distinct and differentially regulated. J. Virol. 70, 8719–8726.

    39. Yu, X., Mertz, J.E., 2003. Distinct modes of regulation of transcription of hepatitis b virus by the nuclear receptors hnf4alpha and coup-tf1. J. Virol. 77, 2489–2499.

    40. Zhang, W., Chen, J., Wu, M., Zhang, X., Zhang, M., Yue, L., Li, Y., Liu, J., Li, B., Shen, F., Wang, Y., Bai, L., Protzer, U., Levrero, M., Yuan, Z., 2017. Prmt5 restricts hepatitis b virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic rna encapsidation. Hepatology 66, 398–415.

    41. Zhao, K., Liu, S., Chen, Y., Yao, Y., Zhou, M., Yuan, Y., Wang, Y., Pei, R., Chen, J., Hu, X., Zhou, Y., Zhao, H., Lu, M., Wu, C., Chen, X., 2018. Upregulation of hbv transcription by sodium taurocholate cotransporting polypeptide at the postentry step is inhibited by the entry inhibitor myrcludex b. Emerg. Microb. Infect. 7, 186.

    42. Zhou, M., Zhao, K., Yao, Y., Yuan, Y., Pei, R., Wang, Y., Chen, J., Hu, X., Zhou, Y., Chen, X., Wu, C., 2017. Productive hbv infection of well-differentiated, hntcpexpressing human hepatoma-derived (huh7) cells. Virol. Sin. 32, 465–475.

  • 加载中

Article Metrics

Article views(304) PDF downloads(6) Cited by()

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection

      Corresponding author: Jing Liu, liujing212@fudan.edu.cn
      Corresponding author: Youhua Xie, yhxie@fudan.edu.cn
    • a Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai, 200032, China;

    Abstract: Hepatitis B virus (HBV) is a primary cause of chronic liver diseases in humans. HBV infection exhibits strict host and tissue tropism. HBV core promoter (Cp) drives transcription of pregenomic RNA (pgRNA) and plays a key role in the viral life cycle. Hepatocyte nuclear factor 4α (HNF4α) acts as a major transcriptional factor that stimulates Cp. In this work, we reported that BEL7404 cell line displayed a high efficiency of DNA transfection and high levels of HBV antigen expression after transfection of HBV replicons without prominent viral replication. The introduction of exogenous HNF4α and human sodium taurocholate cotransporting polypeptide (hNTCP) expression into BEL7404 made it permissive for HBV replication and susceptible to HBV infection. BEL7404-derived cell lines with induced HBV permissiveness and susceptibility were constructed by stable co-transfection of hNTCP and Tet-inducible HNF4α followed by limiting dilution cloning. HBV replication in such cells was sensitive to inhibition by nucleotide analog tenofovir, while the infection was inhibited by HBV entry inhibitors. This cell culture system provides a new and additional tool for the study of HBV replication and infection as well as the characterization of antiviral agents.

    Reference (42) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return