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.
Bibliographical noteFunding Information:
This study was supported by the National Research Foundation of Korea ( MSIT 2017M3A9G6068246 and NRF-2014R1A2A1A11052535 ) and the Gyeonggi Provincial Government.
We would like to thank Dieter Glebe (Giessen University) for providing antibodies and plasmids, and Gil-Je Lee and Ernest Jonathan Cechetto (Perkin-Elmer) for technical assistance in handling microscopy samples and for providing support for the phenotypic analysis of HBV cluster formation. We would like to thank Eunjin Do (Yonsei University) for technical assistance in cccDNA Southern blotting, Stephan Urban and Yi Ni (Heidelberg University) for providing protocols and technical support for the isolation of infectious virions from HepAD38 cells, and Inyoung Kim (Blickfang designstudio) for graphic design support. We thank Heinz. We thank the National Research Foundation of Korea (MSIT and NRF) and the Gyeonggi Provincial Government for financial support.
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