乙型肝炎病毒（HBV）基因组再循环和从头继发感染事件维持

StephenW

J Hepatol. 2018 Aug 21. pii: S0168-8278(18)32294-3. doi: 10.1016/j.jhep.2018.08.012. [Epub ahead of print]
Hepatitis B virus (HBV) genome recycling and de novo secondary infection events maintain stable cccDNA levels.
Ko C1, Chakraborty A2, Chou WM1, Hasreiter J1, Wettengel JM1, Stadler D1, Bester R1, Asen T1, Zhang K1, Wisskirchen K1, McKeating JA3, Ryu WS4, Protzer U5.
Author information

1
    Institute of Virology, Technische Universität München / Helmholtz Zentrum München, Munich, Germany.
2
    Institute of Virology, Technische Universität München / Helmholtz Zentrum München, Munich, Germany; Technische Universität München, Institute for Advanced Study, Munich, Germany.
3
    Technische Universität München, Institute for Advanced Study, Munich, Germany; Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
4
    Department of Biochemistry, Yonsei University, Seoul, Korea.
5
    Institute of Virology, Technische Universität München / Helmholtz Zentrum München, Munich, Germany; Technische Universität München, Institute for Advanced Study, Munich, Germany; German Center for Infection Research (DZIF), Munich partner site, Munich, Germany. Electronic address: [email protected].

Abstract
BACKGROUND & AIMS:

Several steps of the HBV life-cycle including particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes remain obscure due to a lack of robust in vitro infection models. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture.
METHODS:

We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life-cycle and characterized its replication kinetics and dynamics over 6 week-infection.
RESULTS:

HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 hours post-infection and increased until 3 days post infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed this kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5 - 12 copies per infected cell. Newly produced relaxed circular (rc) DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of rcDNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics showing a slow decay with a half-life of about 40 days.
CONCLUSIONS:

After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents.
LAY SUMMARY:

Using a highly permissive hepatocellular model system, we demonstrate that HBV has a remarkably slow infection kinetics. Establishment of the episomal transcription template and persistence form, so called cccDNA, but also viral transcription and protein expression is protracted. Once established, HBV maintains a stable pool of cccDNA via intracellular recycling of HBV genomes and through infection of naive cells by newly formed virions.

Copyright © 2018. Published by Elsevier B.V.
KEYWORDS:

DMSO; HBV; NTCP; PEG; cccDNA; hepatitis B virus; intracellular recycling; reinfection; replenishment; transmission; virus spread

PMID:
    30142426
DOI:
    10.1016/j.jhep.2018.08.012

StephenW

J Hepatol。 2018年8月21日.pii：S0168-8278（18）32294-3。 doi：10.1016 / j.jhep.2018.08.012。 [提前打印]
乙型肝炎病毒（HBV）基因组再循环和从头继发感染事件维持稳定的cccDNA水平。
Ko C1，Chakraborty A2，Chou WM1，Hasreiter J1，Wettengel JM1，Stadler D1，Bester R1，Asen T1，Zhang K1，Wisskirchen K1，McKeating JA3，Ryu WS4，Protzer U5。
作者信息

1
    德国慕尼黑工业大学/ HelmholtzZentrumMünchen病毒学研究所。
2
    德国慕尼黑工业大学/ HelmholtzZentrumMünchen病毒学研究所;慕尼黑工业大学，德国慕尼黑高级研究所。
3
    德国慕尼黑高等研究院慕尼黑工业大学;英国牛津大学牛津大学纳菲尔德医学系。
4
    韩国首尔延世大学生物化学系。
五
    德国慕尼黑工业大学/ HelmholtzZentrumMünchen病毒学研究所;德国慕尼黑高等研究院慕尼黑工业大学;德国感染研究中心（DZIF），慕尼黑合作伙伴网站，德国慕尼黑。电子地址：[email protected]。

抽象
背景与目的：

HBV生命周期的几个步骤包括粒子进入，共价闭合环状（ccc）DNA的形成和维持，基因表达的动力学和病毒传播途径由于缺乏稳健的体外感染模型而仍然模糊不清。本研究旨在研究长期培养过程中的感染动力学和cccDNA动态。
方法：

我们选择了一种高度宽容的HepG2-NTCP-K7细胞克隆，该细胞克隆经过工程改造以表达牛磺胆酸钠共转运多肽（NTCP），该多肽支持完整的HBV生命周期，并在6周感染过程中表征其复制动力学和动力学。
结果：

HBV感染动力学表明感染过程缓慢。仅在感染后24小时检测到核cccDNA，并且在感染后3天（dpi）增加核cccDNA。病毒RNA从3 dpi增加到6 dpi达到平台期。 HBV蛋白水平遵循这一动力学，HBx水平首先达到稳定水平。在整个45天的研究期间，cccDNA水平适度增加，每个感染细胞5-12个拷贝。将衣壳内新产生的松弛环状（rc）DNA重新导入细胞核并补充cccDNA库。除了HBV基因组的细胞内循环外，继发性新发感染事件导致cccDNA形成。通过核苷类似物处理感染细胞抑制rcDNA形成使我们能够测量cccDNA动力学，显示缓慢衰减，半衰期为约40天。
结论：

在慢速感染过程后，HBV通过HBV基因组的细胞内循环和二次感染维持稳定的cccDNA库。我们的研究结果为HBV感染的动态提供了重要的见解，并支持未来设计和评估新的抗病毒药物。
LAY概要：

使用高度宽容的肝细胞模型系统，我们证明HBV具有非常缓慢的感染动力学。建立游离转录模板和持久性形式，即所谓的cccDNA，也是病毒转录和蛋白质表达的延长。一旦建立，HBV通过HBV基因组的细胞内循环和通过新形成的病毒粒子感染幼稚细胞来维持稳定的cccDNA库。

版权所有©2018。Elsevier B.V.
关键词：

DMSO; HBV; NTCP; PEG; cccDNA的;乙型肝炎病毒;细胞内循环;再感染;补充;传输;病毒传播

结论：
    30142426
DOI：
    10.1016 / j.jhep.2018.08.012