Applied Molecular Virology Laboratory, Institut Pasteur Korea, Seongnam-si, South Korea.
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.
J Hepatol. 2019 Aug;71(2):289-300. doi: 10.1016/j.jhep.2019.04.010. Epub 2019 May 8.
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.
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.
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.
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.
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.
乙型肝炎病毒（HBV）在感染的肝脏中传播的同时也会被分泌到血液中。HBV 易感的体外感染模型不能有效地扩增病毒后代或支持细胞间传播。我们试图建立一种从临床标本中扩增感染性 HBV 的细胞培养系统。
选择一种能够产生高滴度感染性后代的 HBV 易感牛磺胆酸钠共转运多肽过表达 HepG2 细胞克隆（HepG2-NTCPsec+）。通过天然凝胶电泳和电子显微镜对分泌的 HBV 后代进行表征。进行比较 RNA-seq 转录组学以定量检测宿主前病毒和限制因子的表达。使用 HBV 进入或复制抑制剂、在报告细胞中观察病毒细胞间传播、以及最近邻感染确定来评估病毒传播途径。分析 HBV 基因型 B-D 的扩增动力学。
感染的 HepG2-NTCPsec+细胞分泌高水平的大 HBV 表面蛋白包裹的感染性 HBV 后代，在电子显微镜下具有典型的外观。RNA-seq 转录组学显示，HBV 在 HepG2-NTCPsec+中不会诱导明显的基因表达变化，然而，有利于 HBV 扩增的转录因子的表达比在不太允许的 HepG2-NTCPsec-中更强。用含有 HBV 的患者血清接种后，受感染细胞的比例最初从 10%增加到 70%，通过细胞间传播到相邻细胞，并且有效地分泌了病毒后代和抗原。根据病毒来源，HepG2-NTCPsec+支持高达 1300 倍的 HBV 基因组净扩增。进入和复制抑制剂可消除病毒传播和扩增；抑制剂停用后观察到病毒反弹。
新型 HepG2-NTCPsec+细胞能够有效地支持 HBV 的完整生命周期，以及源自患者或细胞培养的 HBV 的长期病毒传播和扩增，类似于 HBV 感染患者的相关特征。
目前可用的实验室系统无法再现 HBV 通过感染的肝脏传播并释放到血液中的动态过程。我们开发了一种缓慢分裂的肝源性细胞系，该细胞系在接种患者或细胞培养衍生的 HBV 时会增殖感染性病毒颗粒。这种新的感染模型可以通过预先测量患者的 HBV 株对特定抗病毒药物的敏感性来改善治疗效果。
