乙型肝炎病毒基因组再循环和从头继发感染事件维持稳定的cc

StephenW

Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels
Chunkyu Ko
, Anindita Chakraborty
, Wen-Min Chou
, Julia Hasreiter
, Jochen M. Wettengel
, Daniela Stadler
, Romina Bester
, Theresa Asen
, Ke Zhang
, Karin Wisskirchen
, Jane A. McKeating
, Wang-Shick Ryu
, Ulrike Protzer'Correspondence information about the author Ulrike ProtzerEmail the author Ulrike Protzer
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DOI: https://doi.org/10.1016/j.jhep.2018.08.012 |
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Highlights

    •Studying HBV has been limited by the availability of in vitro and in vivo models.
    •A selected HepG2 cell clone expressing NTCP supports long-term HBV infection.
    •HBV has slow infection kinetics requiring 3 days for full establishment of infection.
    •HBV establishes 1–9 copies of cccDNA per cell which have an estimated half-life of 40 days.
    •cccDNA levels remain stable by intracellular genome recycling and secondary infection.

Background & Aims

Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. 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. We characterized the replication kinetics and dynamics of HBV over six weeks of infection.
Results

HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h 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 similar 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 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 relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly 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 unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.
Keywords:
HBV, Hepatitis B virus, cccDNA, NTCP, Replenishment, Viral spread, Transmission, Intracellular recycling

StephenW

乙型肝炎病毒基因组再循环和从头继发感染事件维持稳定的cccDNA水平
Chunkyu Ko
，Anindita Chakraborty
，周文敏
，Julia Hasreiter
，Jochen M. Wettengel
，Daniela Stadler
，罗米娜贝斯特
，特蕾莎阿森
，柯珂
，Karin Wisskirchen
，Jane A. McKeating
，Wang-Shick Ryu
，Ulrike Protzer'关于作者Ulrike ProtzerEmail的作者Ulrike Protzer的相关信息
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DOI：https：//doi.org/10.1016/j.jhep.2018.08.012 |
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强调

    •研究HBV受到体外和体内模型的限制。
    •表达NTCP的选定HepG2细胞克隆支持长期HBV感染。
    •HBV感染动力学缓慢，需要3天才能完全感染。
    •HBV每个细胞建立1-9个cccDNA拷贝，估计半衰期为40天。
    •细胞内基因组循环和继发感染使cccDNA水平保持稳定。

背景与目的

由于缺乏稳健的体外感染模型，HBV生命周期中的几个步骤仍然模糊不清。这些步骤包括粒子进入，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。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，用于表达支持完整HBV生命周期的牛磺胆酸钠共转运多肽（NTCP）。我们描述了感染六周后HBV的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。核cccDNA仅在感染后24小时检测到并且增加至感染后3天（dpi）。病毒RNA从3dpi增加到6dpi达到平台期。 HBV蛋白水平遵循相似的动力学，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松弛的环状DNA形成使我们能够测量cccDNA动力学。 HBV cccDNA缓慢衰减，半衰期约为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。
放置摘要

使用旨在支持病毒生长的独特肝细胞模型系统，我们证明乙型肝炎病毒（HBV）具有显着缓慢的感染动力学。游离转录模板和病毒的持久形式的建立，所谓的共价闭合环状DNA，以及病毒转录和蛋白质表达都需要很长时间。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共价闭合环状DNA库。
关键词：
乙型肝炎病毒，乙型肝炎病毒，cccDNA，NTCP，补给，病毒传播，传播，细胞内回收

StephenW

Ulrike Protzer是一位备受尊敬的德国HBV研究员。 在这个试管模型中估计cccDNA的半衰期是40天。

The mechanism of cccDNA
replenishment could be explained by: (i) secondary infection via extracellular particles,
(ii) direct cell-to-cell transmission, or (iii) intracellular trafficking of mature, HBV rcDNAcontaining
capsids back to the nucleus resulting in nuclear reimport of newly formed rcDNA.
This cycle can be prevented by inhibitors of reverse transcription or by core protein allosteric modulators that have
not only been reported to inhibit formation of rcDNA-containing capsids but also to
perturb cccDNA biosynthesis (unpublished data and [10]).
An alternative pathway to maintain the cccDNA pool is via secondary infection of naïve
cells or pre-infected cells by extracellular virus particles. Enveloped viruses can initiate
new infection events via the transfer of extracellular particles to naïve target cells or
via direct cell-to-cell contacts [11]. Cell-free spread of extracellular progeny virus
allows transition throughout the body to infect distant cells or organs or even a new
host. It can be prevented by neutralizing antibodies or entry inhibitors targeting virus receptor
interaction. In contrast, direct cell-to-cell transmission routes enable a virus
to evade neutralizing antibodies or complement and are resistant to entry inhibitors.

cccDNA的机制
补充可以解释为：（i）通过细胞外颗粒的继发感染，
（ii）直接细胞间传播，或（iii）含有HBV rcDNA的成熟细胞内运输
衣壳体返回细胞核，导致新形成的核重新进入 rcDNA。
可以防止这种循环 通过逆转录抑制剂或具有核心蛋白质的变构调节剂
据报道，不仅可以抑制含有rcDNA的衣壳的形成
干扰cccDNA生物合成（未发表的数据和[10]）。
维持cccDNA库的另一种途径是通过幼稚的继发感染
细胞外病毒颗粒细胞或预先感染的细胞。包含的病毒可以启动
新的感染事件通过细胞外颗粒转移到幼稚靶细胞或
通过直接的细胞间接触[11]。细胞外后代病毒的无细胞传播
允许在整个身体的过渡感染远处的细胞或器官甚至新的
主办。它可以通过中和抗体或靶向病毒受体的进入抑制剂来预防
相互作用。相反，直接的细胞到细胞传播途径使病毒成为可能
逃避中和抗体或补体，并对进入抑制剂具有抗性。

newchinabok

这个发现很重要

newchinabok

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，希望核衣壳发挥作用

newchinabok

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，希望核衣壳发挥作用

SK蜗牛

cccdna的半衰期只有40天啊？