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

Research output: Contribution to journalArticle

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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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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, Kwang Hyub ; 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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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.",
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 Ryu, {Wang Shick} and Kim, {Jung Hee} and Yoon, {Seung Kew} and Park, {Jun Yong} and Ahn, {Sang Hoon} and Han, {Kwang Hyub} 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, WS, Kim, JH, Yoon, SK, Park, JY, Ahn, SH, Han, KH, 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, Kwang Hyub; Gerlich, Wolfram Hubert; Windisch, Marc Peter.

In: Journal of Hepatology, Vol. 71, No. 2, 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, Kwang Hyub

AU - Gerlich, Wolfram Hubert

AU - Windisch, Marc Peter

PY - 2019/8

Y1 - 2019/8

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.

AB - 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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