从头合成乙肝病毒核衣壳对于cccDNA的维持和转录调控是必不

StephenW

D e novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA
Thomas Tu  1   2 , Benno Zehnder  1 , Bingqian Qu  1   3 , Stephan Urban  1   3
Affiliations
Affiliations

    1
    Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, Germany.
    2
    Storr Liver Centre, Westmead Clinical School and Westmead Institute for Medical Research, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia.
    3
    German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany.

    PMID: 33385130 PMCID: PMC7771110 DOI: 10.1016/j.jhepr.2020.100195

Abstract

Background & aims: Chronic HBV infection cannot be cured by current therapeutics owing to their limited ability to reduce covalently closed circular (ccc)DNA levels in the livers of infected individuals. Therefore, greater understanding of the molecular determinants of cccDNA formation and persistence is required. One key issue is the extent to which de novo nucleocapsid-mediated replenishment (reimport) contributes to cccDNA levels in an infected hepatocyte.

Methods: We engineered an infectious HBV mutant with a genome encoding a stop codon at position T67 in the HBV core open reading frame (ΔHBc HBV). Importantly, ΔHBc HBV virions cannot initiate nucleocapsid synthesis upon infection. Long-term in vitro HBV infection markers were followed for up for 9 weeks in HepG2-NTCP cells (A3 clone) and HBV DNA was quantified using a newly-developed, highly-precise PCR assay (cccDNA inversion quantitative PCR).

Results: ΔHBc and wild-type (WT) HBV resulted in comparable expression of HBV surface antigen (HBsAg), which could be blocked using the entry inhibitor Myrcludex B, confirming bona fide infection via the receptor sodium taurocholate cotransporting polypeptide (NTCP). In primary human hepatocytes, Huh7-NTCP, HepG2-NTCP, and HepaRG-NTCP cells, comparable copy numbers of cccDNA were formed. cccDNA levels, transcription of viral RNA, and HBsAg secretion remained comparably stable in WT and ΔHBc HBV-infected cells for at least 9 weeks.

Conclusions: Our results imply that de novo synthesised HBc plays a minor role in transcriptional regulation of cccDNA. Importantly, we show that initially-formed cccDNA is stable in hepatocytes without requiring continuous replenishment in in vitro infection systems and contribution from de novo DNA-containing nucleocapsids is not required. Thus, short-term therapeutic targeting of capsid-reimport is likely an inefficient strategy in eliminating cccDNA in chronically infected hepatocytes.

Lay summary: The hepatitis B virus can maintain itself in the liver for a patient's lifetime, causing liver injury and cancer. We have clarified exactly how it maintains itself in an infected cell. This now means we have a better idea at how to target the virus and cure a chronic infection.

Keywords: ALT, alanine aminotransferase; Antivirals; Bulevirtide; CIs, capsid inhibitors; Capsid inhibitors; Core protein; Covalently closed circular DNA; DHBV, duck hepatitis B virus; HBV DNA integration; HBV persistence; HBV, hepatitis B virus; HBcAg; HBsAg, hepatitis B virus surface antigen; Hepcludex; Myrcludex B; NC, naked capsids; NTCP, sodium taurocholate cotransporting polypeptide; NUCs, nucleos(t)ide analogues; ORF, open reading frame; PEG, polyethylene glycol; PHH, primary human hepatocytes; SN, supernatant; VP, virions; WT, wild-type; cccDNA, covalently closed circular DNA; dpi, days post inoculation; mge, multiplicity of genomic equivalent; pgRNA, pregenomic RNA; rcDNA, relaxed circular DNA; vge, viral genome equivalents.

© 2020 The Authors.
Conflict of interest statement

S.U. is co-applicant and co-inventor on patents protecting HBV preS-derived lipopeptides (Myrcludex B) for the use of HBV/HDV entry inhibitors. The other authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details.

StephenW

从头合成乙肝病毒核衣壳对于cccDNA的维持和转录调控是必不可少的
Thomas Tu 1 2，Benno Zehnder 1，Bingqian Qu 1 3，Stephan Urban 1 3
隶属关系
隶属关系

    1个
    德国海德堡海德堡大学医院分子病毒学传染病科。
    2
    澳大利亚新南威尔士州悉尼大学，韦斯特米德临床医学院斯托尔肝中心，韦斯特米德医学研究学院，医学与卫生学院韦斯特米德医学研究所。
    3
    德国感染研究中心（DZIF），海德堡合作伙伴站点，德国海德堡。

    PMID：33385130 PMCID：PMC7771110 DOI：10.1016 / j.jhepr.2020.100195

抽象

背景与目的：由于慢性乙型肝炎病毒降低感染个体肝脏中共价闭合环（ccc）DNA水平的能力有限，因此目前的治疗方法无法治愈。因此，需要对cccDNA形成和持久性的分子决定因素有更多的了解。一个关键问题是从头开始的核衣壳介导的补充（再输入）在多大程度上影响了受感染肝细胞中的cccDNA水平。

方法：我们设计了一种感染性HBV突变体，其基因组编码了HBV核心开放阅读框（ΔHBcHBV）中T67位置的终止密码子。重要的是，ΔHBcHBV病毒粒子在感染后不能启动核衣壳合成。在HepG2-NTCP细胞（A3克隆）中对长期体外HBV感染标记物进行了长达9周的随访，并使用新开发的高精度PCR测定法（cccDNA反向定量PCR）对HBV DNA进行了定量。

结果：ΔHBc和野生型（WT）HBV导致了HBV表面抗原（HBsAg）的可比表达，这可以通过进入抑制剂Myrcludex B阻断，从而证实通过牛磺胆酸钠共转运多肽（NTCP）受体进行了真正的感染。在原代人肝细胞Huh7-NTCP，HepG2-NTCP和HepaRG-NTCP细胞中，形成了相当数量的cccDNA拷贝数。在WT和ΔHBcHBV感染的细胞中，cccDNA水平，病毒RNA的转录和HBsAg分泌保持相对稳定至少9周。

结论：我们的结果表明，从头合成的HBc在cccDNA转录调控中起次要作用。重要的是，我们证明了最初形成的cccDNA在肝细胞中是稳定的，不需要在体外​​感染系统中进行连续补充，并且不需要包含从头DNA的核衣壳的贡献。因此，衣壳再导入的短期治疗靶向可能是消除慢性感染的肝细胞中cccDNA的无效策略。

摘要：乙型肝炎病毒可以在患者的一生中维持在肝脏中，导致肝损伤和癌症。我们已经明确阐明了它如何在感染的细胞中自我维持。现在，这意味着我们对如何针对病毒和治愈慢性感染有了更好的认识。

关键词：ALT，丙氨酸转氨酶；抗病毒药布列韦肽; CI，衣壳抑制剂；衣壳抑制剂；核心蛋白；共价封闭的环状DNA； DHBV，鸭乙肝病毒； HBV DNA整合； HBV持续性； HBV，乙肝病毒；乙肝表面抗原HBsAg，乙肝病毒表面抗原； Hepcludex; Myrcludex B; NC，裸衣壳； NTCP，牛磺胆酸钠共转运多肽； NUCs，核苷（核苷酸）类似物； ORF，开放阅读框； PEG，聚乙二醇； PHH，原代人肝细胞； SN，上清液；副总裁，病毒粒子； WT，野生型； cccDNA，共价闭合的环状DNA； dpi，接种后天数； mge，基因组当量的多重性； pgRNA，前基因组RNA； rcDNA，松弛环状DNA； vge，病毒基因组等效物。

©2020作者。
利益冲突声明

S.U.是保护使用HBV / HDV进入抑制剂的HBV preS衍生脂肽（Myrcludex B）的专利的共同申请人和共同发明人。其他作者声明没有与此工作有关的利益冲突。有关更多详细信息，请参阅随附的ICMJE披露表格。

StephenW

https://www.jhep-reports.eu/article/S2589-5559(20)30129-4/pdf

newchinabok

吾辈今生能不能看见功能性治愈这一天，难说

windu

从理论上否定了核衣壳的前途？

windu

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