한빛사 논문
Alexander König1, a, Jaewon Yang1, a, Eunji Jo1, Kyu Ho Paul Park1, Hyun Kim1, 2, 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 Windisch1,2,*
1 Applied Molecular Virology Laboratory, Institut Pasteur Korea, Seongnam-si, South Korea
2 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, Ohio, 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
*Corresponding author : Marc Peter Windisch
aAlexander König and Jaewon Yang contributed equally to this work
Abstract
Background & Aims
Hepatitis B virus (HBV) spreads through the infected liver paralleled by secretion 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
Selection of an HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny virus. Secreted HBV progeny was characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics to quantify the expression of host proviral and restriction factors. Evaluation of viral spread routes using HBV entry- or replication inhibitors; visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Analysis of amplification kinetics of HBV genotypes B-D from 7 patients or cell culture for 8 weeks of infection.
Results
Infected HepG2-NTCPsec+ secreted high levels of LHBs-enveloped infectious HBV progeny with typical appearance in EM. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were stronger expressed than in less permissive HepG2-NTCPsec-. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from initially 10% 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 virus source. 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 lifecycle, 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 unsuitable 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 sensitivity of the patient’s HBV strain to the chosen antiviral drugs.
Keywords : complete HBV lifecycle; HepG2-NTCP; patient-derived HBV; drug sensitivity; kinetics of antigen, virion secretion, cccDNA accumulation; HBV doubling time
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