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, KwangHyub Han, Wolfram Hubert Gerlich, Marc Peter Windisch

Research output: Contribution to journalArticle

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 1

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Hep G2 Cells
Hepatitis B virus
Infection
Hepatovirus
Cell Culture Techniques
Liver
Electron Microscopy
RNA
Virus Internalization
Viral Antigens
Virus Replication
Life Cycle Stages

All Science Journal Classification (ASJC) codes

  • Hepatology

Cite this

König, Alexander ; Yang, Jaewon ; Jo, Eunji ; Park, Kyu Ho Paul ; Kim, Hyun ; Than, Thoa Thi ; Song, Xiyong ; Qi, Xiaoxuan ; Dai, Xinghong ; Park, Soonju ; Shum, David ; Ryu, Wang-Shick ; Kim, Jung Hee ; Yoon, Seung Kew ; Park, Jun Yong ; Ahn, Sang Hoon ; Han, KwangHyub ; Gerlich, Wolfram Hubert ; Windisch, Marc Peter. / Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells. In: Journal of Hepatology. 2019 ; Vol. 71, No. 2. pp. 289-300.
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author = "Alexander K{\"o}nig and Jaewon Yang and Eunji Jo and Park, {Kyu Ho Paul} and Hyun Kim and Than, {Thoa Thi} and Xiyong Song and Xiaoxuan Qi and Xinghong Dai and Soonju Park and David Shum and Wang-Shick Ryu and Kim, {Jung Hee} and Yoon, {Seung Kew} and Park, {Jun Yong} and Ahn, {Sang Hoon} and KwangHyub Han and Gerlich, {Wolfram Hubert} and Windisch, {Marc Peter}",
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König, A, Yang, J, Jo, E, Park, KHP, Kim, H, Than, TT, Song, X, Qi, X, Dai, X, Park, S, Shum, D, Ryu, W-S, Kim, JH, Yoon, SK, Park, JY, Ahn, SH, Han, K, Gerlich, WH & Windisch, MP 2019, 'Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells', Journal of Hepatology, vol. 71, no. 2, pp. 289-300. https://doi.org/10.1016/j.jhep.2019.04.010

Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells. / König, Alexander; Yang, Jaewon; Jo, Eunji; Park, Kyu Ho Paul; Kim, Hyun; Than, Thoa Thi; Song, Xiyong; Qi, Xiaoxuan; Dai, Xinghong; Park, Soonju; Shum, David; Ryu, Wang-Shick; Kim, Jung Hee; Yoon, Seung Kew; Park, Jun Yong; Ahn, Sang Hoon; Han, KwangHyub; Gerlich, Wolfram Hubert; Windisch, Marc Peter.

In: Journal of Hepatology, Vol. 71, No. 2, 01.08.2019, p. 289-300.

Research output: Contribution to journalArticle

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T1 - Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells

AU - König, Alexander

AU - Yang, Jaewon

AU - Jo, Eunji

AU - Park, Kyu Ho Paul

AU - Kim, Hyun

AU - Than, Thoa Thi

AU - Song, Xiyong

AU - Qi, Xiaoxuan

AU - Dai, Xinghong

AU - Park, Soonju

AU - Shum, David

AU - Ryu, Wang-Shick

AU - Kim, Jung Hee

AU - Yoon, Seung Kew

AU - Park, Jun Yong

AU - Ahn, Sang Hoon

AU - Han, KwangHyub

AU - Gerlich, Wolfram Hubert

AU - Windisch, Marc Peter

PY - 2019/8/1

Y1 - 2019/8/1

N2 - 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.

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