人肝癌细胞从头乙型肝炎病毒感染中共价闭合环状DNA合成的

StephenW

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Antiviral Res. 2019 Jan 10. pii: S0166-3542(18)30670-3. doi: 10.1016/j.antiviral.2019.01.004. [Epub ahead of print]
Characterization of the molecular events of covalently closed circular DNA synthesis in de novo Hepatitis B virus infection of human hepatoma cells.
Dezhbord M1, Lee S2, Kim W2, Seong BL3, Ryu WS4.
Author information

1
    Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea.
2
    Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea.
3
    Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea. Electronic address: [email protected].
4
    Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea. Electronic address: [email protected].

Abstract

Despite the utmost importance of cccDNA in HBV biology, the mechanism by which cccDNA synthesis is regulated is not completely understood. Here we explored HepG2-NTCP cell line and performed a time-course HBV infection experiment (up to 30 days) to follow the conversion of the input viral DNA into cccDNA. We found that a protein-free RC DNA (PF-RC DNA) become detectable as early as 12 h post infection (hpi) prior to the detection of cccDNA, which become evident only at 2-3 dpi. Intriguingly, the PF-RC DNA detected at 12 hpi was abundantly located in the cytoplasm, implicating that the protein-removal from the input viral DNA takes place in the cytoplasm, perhaps inside the nucleocapsid. Notably, during the early time points of HBV infection, the PF-RC DNA accumulated at significantly higher levels and appeared in a peak followed by a plateau at late time points with dramatically lower levels, implicating the presence of two distinct populations of the PF-RC DNA. Importantly, the PF-RC DNA at earlier peak is entecavir (ETV)-resistant, whereas the PF-RC DNA at posterior days is ETV-sensitive. An interpretation is that the PF-RC DNA at earlier peak represents "input viral DNA" derived from HBV inoculum, whereas the PF-RC DNA at late time points represents the de novo product of the viral reverse transcription. The existence of two populations of the PF-RC DNA having a distinct kinetic profile and ETV-sensitivity implicated that intracellular amplification via the viral reverse transcription greatly contributes to the maintenance of cccDNA pool during HBV infection. As such, we concluded that the cccDNA level is stably maintained by continuing replenishment of cccDNA primarily through intracellular amplification in the HepG2-NTCP cell line.

Copyright © 2019. Published by Elsevier B.V.
KEYWORDS:

HepG2-NTCP cell line; Hepatitis B infection; Time point HBV infection; cccDNA dynamics

PMID:
    30639437
DOI:
    10.1016/j.antiviral.2019.01.004

StephenW

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抗病毒药物2019年1月10日.pii：S0166-3542（18）30670-3。 doi：10.1016 / j.antiviral.2019.01.004。 [提前打印]
人肝癌细胞从头乙型肝炎病毒感染中共价闭合环状DNA合成的分子事件特征。
Dezhbord M1，Lee S2，Kim W2，Seong BL3，Ryu WS4。
作者信息

1
    韩国首尔延世大学生命科学与生物技术学院生物技术系。
2
    韩国首尔延世大学生命科学与生物技术学院生物化学系。
3
    韩国首尔延世大学生命科学与生物技术学院生物技术系。电子地址：[email protected]。
4
    韩国首尔延世大学生命科学与生物技术学院生物化学系。电子地址：[email protected]。

抽象

尽管cccDNA在HBV生物学中至关重要，但cccDNA合成受调控的机制尚不完全清楚。在这里，我们探索了HepG2-NTCP细胞系，并进行了一个时间过程的HBV感染实验（最多30天），以跟踪输入病毒DNA转换为cccDNA。我们发现，在检测到cccDNA之前，早在感染后12小时（hpi）就可检测到无蛋白质的RC DNA（PF-RC DNA），这种情况仅在2-3 dpi时才变得明显。有趣的是，在12 hpi检测到的PF-RC DNA大量位于细胞质中，这意味着从输入的病毒DNA中去除蛋白质发生在细胞质中，可能在核衣壳内部。值得注意的是，在HBV感染的早期时间点，PF-RC DNA在较高水平积累并出现在峰值，随后在晚期时间点达到平台，水平显着降低，表明PF-存在两个不同的群体。 RC DNA。重要的是，早期峰值的PF-RC DNA是恩替卡韦（ETV）抗性，而后日的PF-RC DNA是ETV敏感的。解释是早期峰值的PF-RC DNA代表来自HBV接种物的“输入病毒DNA”，而晚期时间点的PF-RC DNA代表病毒逆转录的从头产物。具有不同动力学特征和ETV敏感性的两个群体的PF-RC DNA的存在暗示通过病毒逆转录的细胞内扩增极大地有助于在HBV感染期间维持cccDNA库。因此，我们得出结论，通过主要通过HepG2-NTCP细胞系中的细胞内扩增继续补充cccDNA来稳定地维持cccDNA水平。

版权所有©2019。Elsevier B.V.
关键词：

HepG2-NTCP细胞系;乙型肝炎感染;时间点HBV感染; cccDNA动力学

结论：
    30639437
DOI：
    10.1016 / j.antiviral.2019.01.004