Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
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DOI：
10.1016/j.jhep.2018.08.012
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发表时间：
2018-12
影响因子：
25.7
通讯作者：
Protzer U
中科院分区：
文献类型：
--
作者：
Ko C;Chakraborty A;Chou WM;Hasreiter J;Wettengel JM;Stadler D;Bester R;Asen T;Zhang K;Wisskirchen K;McKeating JA;Ryu WS;Protzer U
由于缺乏可靠的体外感染模型，HBV生命周期中的几个步骤仍然不清楚。这些步骤包括颗粒进入、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期间的感染动力学和cccDNA动力学。我们选择了一个高度允许的HepG 2-NTCP-K7细胞克隆，该细胞克隆被工程化以表达支持整个HBV生命周期的牛磺胆酸钠共转运多肽（NTCP）。我们的特点是复制动力学和动态的HBV感染超过六周。HBV感染动力学表现为缓慢的感染过程。核cccDNA仅在感染后24小时检测到，并且增加直到感染后3天（dpi）。病毒RNA从3dpi开始增加，在6dpi达到平台。HBV蛋白水平遵循相似的动力学，HBx水平首先达到平台。cccDNA水平在整个45天研究期间适度增加，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基因组的细胞内再循环，继发性从头感染事件导致cccDNA形成。通过核苷类似物处理感染细胞抑制松弛环状DNA形成使我们能够测量cccDNA动力学。HBV cccDNA衰变缓慢，半衰期约为40天。在缓慢的感染过程后，HBV通过HBV基因组的细胞内再循环和继发感染维持稳定的cccDNA库。我们的研究结果为HBV感染的动力学提供了重要的见解，并支持未来新的抗病毒药物的设计和评估。使用一个独特的肝细胞模型系统，旨在支持病毒的生长，我们证明，B肝炎病毒（HBV）具有显着缓慢的感染动力学。附加型转录模板的建立和病毒的持久形式，即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基因组的细胞内再循环和通过新形成的病毒体感染幼稚细胞来维持共价闭合环状DNA的稳定池。
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. We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate cotransporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection. 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. 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. 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.
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影响因子：
7.6
作者：
Liu C;Cai D;Zhang L;Tang W;Yan R;Guo H;Chen X
通讯作者：
Chen X
DOI：
10.1007/978-1-4939-6700-1_6
发表时间：
2017-01-01
期刊：
HEPATITIS B VIRUS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Xia, Yuchen;Stadler, Daniela;Protzer, Ulrike
通讯作者：
Protzer, Ulrike
影响因子：
3.7
作者：
Seeger, Christoph;Mason, William S.
通讯作者：
Mason, William S.
影响因子：
5.4
作者：
Ko, Chunkyu;Shin, Youn-Chul;Ryu, Wang-Shick
通讯作者：
Ryu, Wang-Shick
影响因子：
2.5
作者：
Nikolaou N;Green CJ;Gunn PJ;Hodson L;Tomlinson JW
通讯作者：
Tomlinson JW