Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection.
Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection.
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DOI：
10.1016/j.virs.2022.05.002
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发表时间：
2022-08
影响因子：
5.5
通讯作者：
Xie, Youhua
中科院分区：
文献类型：
--
作者：
Song, Yingying;Shou, Shuyu;Guo, Huimin;Gao, Zixiang;Liu, Nannan;Yang, Yang;Wang, Feifei;Deng, Qiang;Liu, Jing;Xie, Youhua
关键词：
乙型肝炎病毒（HBV）是人类慢性肝病的主要病因。HBV感染表现出严格的宿主和组织趋向性。HBV核心启动子（Cp）驱动基因组前RNA （pgRNA）的转录，在病毒生命周期中起关键作用。肝细胞核因子4α (HNF4α)是刺激Cp的主要转录因子。在这项工作中，我们报道了BEL7404细胞系在转染HBV复制子后表现出高效率的DNA转染和高水平的HBV抗原表达，而没有明显的病毒复制。在BEL7404中引入外源性HNF4α和人牛磺酸胆酸钠共转运多肽（hNTCP）表达，使其允许HBV复制，易受HBV感染。通过稳定共转染hNTCP和tet诱导的HNF4α，然后限制性稀释克隆，构建了具有诱导HBV许可性和易感性的bel7404来源细胞系。这些细胞中的HBV复制对核苷酸类似物替诺福韦的抑制很敏感，而HBV进入抑制剂则能抑制感染。这种细胞培养系统为研究HBV复制和感染以及抗病毒药物的特性提供了一种新的和额外的工具。BEL7404细胞的特点是转染效率高，但不支持典型的HBV复制。BEL7404细胞缺乏内源性HNF4α表达，外源性HNF4α可以挽救典型HBV复制。具有稳定的hNTCP和可诱导的HNF4α表达的BEL7404细胞支持HBV感染和诱导复制。支持HBV感染的bel7404衍生细胞系保持了高转染效率，并允许抗病毒药物测试。
Hepatitis B virus (HBV) is a primary cause of chronic liver diseases in humans. HBV infection exhibits strict host and tissue tropism. HBV core promoter (Cp) drives transcription of pregenomic RNA (pgRNA) and plays a key role in the viral life cycle. Hepatocyte nuclear factor 4α (HNF4α) acts as a major transcriptional factor that stimulates Cp. In this work, we reported that BEL7404 cell line displayed a high efficiency of DNA transfection and high levels of HBV antigen expression after transfection of HBV replicons without prominent viral replication. The introduction of exogenous HNF4α and human sodium taurocholate cotransporting polypeptide (hNTCP) expression into BEL7404 made it permissive for HBV replication and susceptible to HBV infection. BEL7404-derived cell lines with induced HBV permissiveness and susceptibility were constructed by stable co-transfection of hNTCP and Tet-inducible HNF4α followed by limiting dilution cloning. HBV replication in such cells was sensitive to inhibition by nucleotide analog tenofovir, while the infection was inhibited by HBV entry inhibitors. This cell culture system provides a new and additional tool for the study of HBV replication and infection as well as the characterization of antiviral agents. BEL7404 cells are characterized by a high transfection efficiency, but do not support canonical HBV replication. BEL7404 cells lack endogenous HNF4α expression, and exogenous HNF4α rescues canonical HBV replication. BEL7404 cells with stable hNTCP and inducible HNF4α expression support HBV infection and inducible replication. BEL7404-derived cell lines supporting HBV infection retain high transfection efficiencies and allow testing of antivirals.
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