Alexander König1, Jaewon Yang1, Eunji Jo1, Kyu Ho Paul Park1, Hyun Kim2, Thoa Thi Than1, Xiyong Song3, Xiaoxuan Qi3, Xinghong Dai3, Soonju Park4, David Shum4, Wang-Shick Ryu5, Jung-Hee Kim6, Seung Kew Yoon6, Jun Yong Park7, Sang Hoon Ahn7, Kwang-Hyub Han7, Wolfram Hubert Gerlich8, Marc Peter Windisch9. 1. Applied Molecular Virology Laboratory, Institut Pasteur Korea, Seongnam-si, South Korea. 2. Applied Molecular Virology Laboratory, Institut Pasteur Korea, Seongnam-si, South Korea; Division of Bio-Medical Science and Technology, University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon, South Korea. 3. Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. 4. Screening Discovery Platform, Institut Pasteur Korea, Seongnam-si, South Korea. 5. Department of Biochemistry, Yonsei University, Seoul, South Korea. 6. Catholic University Liver Research Center, The Catholic University of Korea, Seoul, South Korea. 7. Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea. 8. Institute of Medical Virology, Justus-Liebig-University, Giessen, Germany. 9. Applied Molecular Virology Laboratory, Institut Pasteur Korea, Seongnam-si, South Korea; Division of Bio-Medical Science and Technology, University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon, South Korea. Electronic address: marc.windisch@ip-korea.org.
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.
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-infectedpatients. 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.
Authors: Jaewon Yang; Alexander König; Soonju Park; Eunji Jo; Pil Soo Sung; Seung Kew Yoon; Eva Zusinaite; Denis Kainov; David Shum; Marc Peter Windisch Journal: JHEP Rep Date: 2021-04-30
Authors: Carla Eller; Laura Heydmann; Che C Colpitts; Houssein El Saghire; Federica Piccioni; Frank Jühling; Karim Majzoub; Caroline Pons; Charlotte Bach; Julie Lucifora; Joachim Lupberger; Michael Nassal; Glenn S Cowley; Naoto Fujiwara; Sen-Yung Hsieh; Yujin Hoshida; Emanuele Felli; Patrick Pessaux; Camille Sureau; Catherine Schuster; David E Root; Eloi R Verrier; Thomas F Baumert Journal: Nat Commun Date: 2020-06-01 Impact factor: 17.694