neddylation 在从 cccDNA 转录乙型肝炎病毒 RNA 和产生病毒表面抗

StephenW

neddylation 在从 cccDNA 转录乙型肝炎病毒 RNA 和产生病毒表面抗原中的双重作用
Bingqian Qu 1 2 , Firat Nebioglu 1 3 , Mila M Leuthold 1 , Yi Ni 1 4 , Pascal Mutz 1 3 , Jürgen Beneke 5 , Holger Erfle 5 , Florian W R Vondran 6 7 , Ralf Bartenschlager 1 3 4 , Stephan Urban 1 4
隶属关系
隶属关系

    1
    海德堡大学传染病学系，分子病毒学，德国海德堡。
    2
    德国朗根 Paul Ehrlich 研究所兽医学部。
    3
    德国海德堡德国癌症研究中心 (DKFZ) 病毒相关癌发生部门 (F170)。
    4
    德国感染研究中心 (DZIF)，海德堡合作伙伴网站，德国海德堡。
    5
    海德堡大学分子和细胞生物系统定量分析高级生物筛选设施中心 (BioQuant)，海德堡，德国。
    6
    德国感染研究中心 (DZIF)，汉诺威-布伦瑞克合作伙伴网站，汉诺威，德国。
    7
    再生医学和实验外科 (ReMediES)，汉诺威医学院普通、内脏和移植外科，德国汉诺威。

    PMID：36124123 PMCID：PMC9482114 DOI：10.1016/j.jhepr.2022.100551

抽象的

背景和目的：HBV DNA 的持久性是通过游离型共价闭合环状 (ccc) DNA 储存库和 HBV DNA 片段的基因组整合来维持的。虽然 cccDNA 转录受 Cullin4A-DDB1-HBx 介导的 SMC5/6 复合物降解的调节，但整合体中的 HBsAg 表达在很大程度上与 SMC5/6 无关。抑制 Cullin-RING 泛素连接酶的 neddylation 会损害底物的降解。在这里，我们显示通过小干扰 (si) RNA 或药物 MLN4924 (pevonedistat) 靶向 neddylation 途径成分可抑制 cccDNA 和整合体中 HBV 蛋白的表达。

方法：进行了靶向分泌途径调节因子和neddylation基因的siRNA筛选。在感染和整合模型中评估了 MLN4924 的活性。反式互补分析用于研究 cccDNA 驱动的表达中的 HBx 功能。

结果：siRNA 筛选揭示了转录后促进 HBsAg 产生的 neddylation 途径成分（Nedd8、Ube2m）。同样，MLN4924 抑制由整合体编码的 HBsAg 的产生并降低细胞内 HBsAg 水平，与 HBx 无关。 MLN4924 还在三种感染模型中显着抑制 cccDNA 转录。使用 HBV 诱导细胞系 HepAD38 作为模型，我们验证了 MLN4924 对 cccDNA 和整合体的双重作用，在 42 天的治疗期间持续抑制 HBV 标志物。

结论： cccDNA 储存库的转录和病毒 DNA 的基因组整合都需要 Neddylation。因此，阻断 neddylation 可能为慢性乙型肝炎的功能性治愈提供一种有吸引力的方法。

总结：目前对慢性乙型肝炎的治疗很少能够引起功能性治愈。这部分是因为宿主细胞核中存在环状病毒 DNA 库，以及整合到宿主基因组中的病毒 DNA 片段。在这里，我们展示了一种称为 neddylation 的宿主生物途径可能在感染和病毒 DNA 整合中起关键作用。抑制这一途径可以为慢性乙型肝炎患者带来治疗希望。

关键词：DDB1，DNA损伤结合蛋白1；乙肝表面抗原； HBsAg，乙型肝炎病毒表面抗原； HBx; HBx，乙型肝炎病毒X蛋白； MLN4924; NAE1，NEDD8激活酶E1亚基1； NEDD8，神经前体细胞表达，发育下调 8； PHHs，原代人肝细胞； SMC6; SVP，亚病毒颗粒； Smc5/6，染色体 5/6 的结构维护； WT，野生型； cccDNA； cccDNA，共价闭合环状 DNA；整合体；内化； pgRNA，前基因组 RNA； siRNA，小干扰RNA；转录。

© 2022 作者。
利益冲突声明

Stephan Urban 是保护 HBV preS1 衍生脂肽（Myrcludex B/Bulevirtide/Hepcludex）专利的共同发明人和申请人。所有其他作者声明没有利益冲突。请参阅随附的 ICMJE 披露表格了解更多详情。

StephenW

Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen
Bingqian Qu  1   2 , Firat Nebioglu  1   3 , Mila M Leuthold  1 , Yi Ni  1   4 , Pascal Mutz  1   3 , Jürgen Beneke  5 , Holger Erfle  5 , Florian W R Vondran  6   7 , Ralf Bartenschlager  1   3   4 , Stephan Urban  1   4
Affiliations
Affiliations

    1
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
    2
    Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany.
    3
    Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany.
    4
    German Center for Infection Research (DZIF), Heidelberg Partner site, Heidelberg, Germany.
    5
    Advanced Biological Screening Facility Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), Heidelberg University, Heidelberg, Germany.
    6
    German Center for Infection Research (DZIF), Hannover-Braunschweig Partner site, Hannover, Germany.
    7
    Regenerative Medicine and Experimental Surgery (ReMediES), Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.

    PMID: 36124123 PMCID: PMC9482114 DOI: 10.1016/j.jhepr.2022.100551

Abstract

Background & aims: HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA reservoir and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting neddylation of Cullin-RING ubiquitin ligases impairs degradation of substrates. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants.

Methods: An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA-driven expression.

Results: siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model, we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment.

Conclusions: Neddylation is required both for transcription of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B.

Lay summary: Current treatments for chronic hepatitis B are rarely able to induce a functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.

Keywords: DDB1, DNA damage-binding protein 1; HBsAg; HBsAg, hepatitis B virus surface antigen; HBx; HBx, hepatitis B virus X protein; MLN4924; NAE1, NEDD8-activating enzyme E1 subunit 1; NEDD8, neural precursor cell expressed, developmentally downregulated 8; PHHs, primary human hepatocytes; SMC6; SVP, subviral particles; Smc5/6, structural maintenance of chromosomes 5/6; WT, wild-type; cccDNA; cccDNA, covalently closed circular DNA; integrants; neddylation; pgRNA, pregenomic RNA; siRNA, small-interfering RNA; transcription.

© 2022 The Author(s).
Conflict of interest statement

Stephan Urban is co-inventor and applicant on patents protecting HBV preS1-derived lipopeptides (Myrcludex B/Bulevirtide/Hepcludex). All other authors declare no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details.

StephenW

http://www.jhep-reports.eu/article/S2589555922001239/pdf

StephenW

讨论摘录：
HBV 整合导致受感染肝细胞的基因组不稳定
细胞，这有助于致癌。 44,45 HBV 整合
化在此过程中起着重要作用，并发生在后期
感染。 46 虽然 HBV 复制不直接需要，但
整合子通常编码 HBx 宿主嵌合基因和所有 HBV
真正的启动子控制下的包膜蛋白。 47,48
最近的临床发现揭示了克隆扩展的整合体
作为 HBeAg 阴性患者 HBsAg 的主要来源，
新药开发面临的挑战。 22,25,46 此外，
高水平的 HBsAg 表达是 im 不足的关键驱动因素
HBV 感染的免疫控制，因此，
治愈性治疗方法。
MLN4924 是一种用于治疗固体肿瘤的化疗药物
痛风和血液系统恶性肿瘤。表现得很好
在 II/III 期临床试验中的耐受性，并已获得突破
通过状态。作为一种抗病毒药物，它的使用可能会受到限制，
然而，cccDNA 中的 HBsAg 快速而显着降低
和整合体，例如结合免疫调节剂，可以
使 MLN4924 成为有吸引力的候选者，尤其是在瞄准时
在引发免疫恢复的导入方案中
系统实现病毒清除。

StephenW

Extracts from Discussion:
HBV integration causes genomic instability in infected hepa-
tocytes, which contributes to carcinogenesis. 44,45 HBV integra-
tion plays an important role in this process and occurs early post
infection. 46 While not directly required for HBV replication,
integrants typically encode HBx-host chimeric genes and all HBV
envelope proteins under control of the authentic promoter.47,48
Recent clinical findings uncovered clonally expanded integrants
as a major source of HBsAg in HBeAg-negative patients, which is
a challenge for the development of novel drugs. 22,25,46 Moreover,
high-level HBsAg expression is a key driver of insufficient im-
mune control of HBV infection and therefore, a major target for
curative therapeutic approaches.
MLN4924 is a chemotherapeutic drug used to treat solid tu-
mours and haematological malignancies. It demonstrated good
tolerability in phase II/III clinical trials and has received break-
through status. As an antiviral drug its use might be limited,
however a fast and profound reduction of HBsAg from cccDNA
and integrants, e.g. combined with immune-modulators, could
make MLN4924 an attractive candidate, especially when aiming
at lead-in regimens that provoke restoration of the immune
system to achieve viral clearance.