Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells

Alexander König, Jaewon Yang, Eunji Jo, Kyu Ho Paul Park, Hyun Kim, Thoa Thi Than, Xiyong Song, Xiaoxuan Qi, Xinghong Dai, Soonju Park, David Shum, Wang Shick Ryu, Jung Hee Kim, Seung Kew Yoon, Jun Yong Park, Sang Hoon Ahn, Kwang Hyub Han, Wolfram Hubert Gerlich, Marc Peter Windisch

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3 Citations (Scopus)

Abstract

Background & Aims: As hepatitis B virus (HBV) spreads through the infected liver it is simultaneously secreted into the blood. HBV-susceptible in vitro infection models do not efficiently amplify viral progeny or support cell-to-cell spread. We sought to establish a cell culture system for the amplification of infectious HBV from clinical specimens. Methods: An HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny was selected. Secreted HBV progeny were characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics was performed to quantify the expression of host proviral and restriction factors. Viral spread routes were evaluated using HBV entry- or replication inhibitors, visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Amplification kinetics of HBV genotypes B-D were analyzed. Results: Infected HepG2-NTCPsec+ secreted high levels of large HBV surface protein-enveloped infectious HBV progeny with typical appearance under electron microscopy. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were more strongly expressed than in less permissive HepG2-NTCPsec−. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from 10% initially to 70% by viral spread to adjacent cells, and viral progeny and antigens were efficiently secreted. HepG2-NTCPsec+ supported up to 1,300-fold net amplification of HBV genomes depending on the source of virus. Viral spread and amplification were abolished by entry and replication inhibitors; viral rebound was observed after inhibitor discontinuation. Conclusions: The novel HepG2-NTCPsec+ cells efficiently support the complete HBV life cycle, long-term viral spread and amplification of HBV derived from patients or cell culture, resembling relevant features of HBV-infected patients. Lay summary: Currently available laboratory systems are unable to reproduce the dynamics of hepatitis B virus (HBV) spread through the infected liver and release into the blood. We developed a slowly dividing liver-derived cell line which multiplies infectious viral particles upon inoculation with patient- or cell culture-derived HBV. This new infection model can improve therapy by measuring, in advance, the sensitivity of a patient's HBV strain to specific antiviral drugs.

Original languageEnglish
Pages (from-to)289-300
Number of pages12
JournalJournal of Hepatology
Volume71
Issue number2
DOIs
Publication statusPublished - 2019 Aug

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All Science Journal Classification (ASJC) codes

  • Hepatology

Cite this

König, A., Yang, J., Jo, E., Park, K. H. P., Kim, H., Than, T. T., Song, X., Qi, X., Dai, X., 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. Journal of Hepatology, 71(2), 289-300. https://doi.org/10.1016/j.jhep.2019.04.010