Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection

Yingying Song; Shuyu Shou; Huimin Guo; Zixiang Gao; Nannan Liu; Yang Yang; Feifei Wang; Qiang Deng; Jing Liu; Youhua Xie

doi:10.1016/j.virs.2022.05.002

August 2022

Yingying Song, Shuyu Shou, Huimin Guo, Zixiang Gao, Nannan Liu, Yang Yang, Feifei Wang, Qiang Deng, Jing Liu and Youhua Xie. Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection[J]. Virologica Sinica, 2022, 37(4): 558-568. doi: 10.1016/j.virs.2022.05.002

Citation: Yingying Song, Shuyu Shou, Huimin Guo, Zixiang Gao, Nannan Liu, Yang Yang, Feifei Wang, Qiang Deng, Jing Liu, Youhua Xie. Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection .VIROLOGICA SINICA, 2022, 37(4) : 558-568. http://dx.doi.org/10.1016/j.virs.2022.05.002

一种可支持乙肝病毒复制和感染的细胞模型的建立及功能研究

宋迎迎 ^a ,
寿澍雨 ^a ,
郭慧敏 ^a,b,e ,
高子翔 ^a ,
刘楠楠 ^a ,
杨阳 ^a ,
王菲菲 ^a ,
邓强 ^a ,
刘晶 ^a,d,, ,
谢幼华 ^a,c,,

复旦大学上海医学院, 基础医学院病原生物学系, 分子病毒学重点实验室

通讯作者： 刘晶, liujing212@fudan.edu.cn; 谢幼华, yhxie@fudan.edu.cn
收稿日期： 2022-01-24
录用日期： 2022-04-18

摘要

乙肝病毒（Hepatitis B virus，HBV）慢性感染与多种肝脏疾病密切相关，并且HBV感染具有严格的宿主和组织特异性。HBV核心启动子Cp启动前基因组RNA （pregenomic RNA，pgRNA）的转录，在病毒生活周期中起着关键作用。肝脏核转录因子HNF4α是促进Cp启动pgRNA转录的主要因子。在本研究中，我们发现BEL7404细胞系具有较好的转染效率，在转染Cp驱动的HBV复制子之后展现较高的HBV抗原表达水平，但未产生明显的病毒复制。BEL7404细胞系中HBV复制缺陷与缺乏内源HNF4α表达有关，在引入外源性HNF4α和人牛磺胆酸钠共转运多肽（hNTCP）表达后，该细胞系可以支持HBV复制和感染。将该细胞系稳定转染hNTCP和四环素（Tet）诱导表达的HNF4α之后，使用限制性稀释法获得了克隆化BEL7404衍生细胞系。在上述衍生细胞系中，HBV的复制对可被HBV聚合酶抑制剂替诺福韦抑制，并且HBV的感染也可被HBV入侵抑制剂阻断。以上基于BEL7404的细胞系统为研究HBV复制和感染以及抗病毒药物的特性提供了新的模型。
- 乙肝病毒(HBV)
- , BEL7404
- , 牛磺胆酸钠共转运多肽(NTCP)
- , 肝脏核转录因子4α(HNF4α)
- , Tet-On
- , 多西环素(DOX)

Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection

Yingying Song ^a ,
Shuyu Shou ^a ,
Huimin Guo ^a,b,e ,
Zixiang Gao ^a ,
Nannan Liu ^a ,
Yang Yang ^a ,
Feifei Wang ^a ,
Qiang Deng ^a ,
Jing Liu ^a,d,, ,
Youhua Xie ^a,c,,

a Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai, 200032, China;

Corresponding author: Jing Liu, liujing212@fudan.edu.cn Youhua Xie, yhxie@fudan.edu.cn
Received Date: 24 January 2022
Accepted Date: 18 April 2022

Abstract

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.
- Hepatitis B virus (HBV)
- , Sodium-taurocholate cotransporting
- , polypeptide (NTCP)
- , BEL7404
- , Hepatocyte nuclear factor 4α (HNF4α)
- , Tetracycline (Tet)-On

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Electronic Supplementary Material

10.1016j.virs.2022.05.002-ESM.docx