通过降解HBx蛋白来沉默HBV附加体：走向功能性治疗？

StephenW

Silencing of the HBV episome through degradation of HBx protein: Towards functional cure?

    Julie Lucifora
    Thomas F. Baumert

Published:November 10, 2020DOI:https://doi.org/10.1016/j.jhep.2020.10.018
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See Article, pages 522–534
Chronic HBV infection is a major cause of advanced liver disease and cancer world-wide, posing a global health challenge. While approved therapies efficiently control HBV viremia, viral elimination is rare. The HBV life cycle displays several unique features that enable the virus to persist in hepatocytes. By replicating its genome exclusively in the neo-formed nucleocapsids, HBV evades recognition by cellular antiviral immune responses. Moreover, HBV also establishes a stable episome in the nucleus of infected cells. This episome, called cccDNA (covalently closed circular DNA) has also been termed the “minichromosome”. Except for the lack of a replication origin, the HBV cccDNA molecule is indeed organized into a chromatin-like structure that displays a typical beads-on-a-string arrangement on electron microscopy.1 The HBV cccDNA serves as a template for transcription of all HBV RNAs by the cellular RNA polymerase II. Modulation of its transcriptional activity results in the recruitment of histones, histone-modifying enzymes and several cellular transcription factors.2,  3,  4,  5,  6 Even though long debated, the role of the viral protein HBx in the control of HBV transcription became clearer in the last decade (for review see7,8). Collectively, several different independent studies have shown that HBx is required for the initiation and maintenance of cccDNA-driven transcription of HBV RNAs.6,9,  10,  11 HBx most likely exerts this key role through different and complementary mechanisms: HBx promotes the degradation of the structural maintenance of chromosomes complex Smc5/6, which was recently identified as a host restriction factor that suppresses RNA transcription from cccDNA.12,  13,  14 It has also been shown that HBx is directly recruited to cccDNA. Absence of HBx results in decreased acetylation of cccDNA-bound histones, impaired recruitment of the transcriptional coactivator p300, and to recruitment of histone deacetylases3 and heterochromatin protein factor 1 (HP1).6 Furthermore, numerous studies (although often performed in less physiological overexpression systems) have reported an effect of HBx on the perturbation of host cellular pathways such as apoptosis, calcium signaling, and the cell cycle, which are associated with liver diseases and hepatocarcinogenesis (for review see8,15). Since HBx plays a central role in HBV infection and is most likely relevant for virus-induced liver disease, it is an interesting target for new therapies to treat chronic HBV infection. This concept is in line with other approaches aiming for “functional cure”, which is characterised by sustained loss of HBsAg (with or without anti-HBs seroconversion) and is associated with improved clinical outcomes.16 In order to identify compounds targeting HBx, Cheng et al.17 screened a library of small molecules for their ability to inhibit HBx expression. Using this approach, they identified dicoumarol, which dose-dependently reduces HBx protein expression without affecting the levels of HBx mRNAs. Among several properties, dicoumarol is a competitive inhibitor of NAD(P)H:quinone oxidoreductase (NQO1).18 Loss- and gain-of -function assays showed that NQO1 binds to HBx and promotes its stability by preventing its degradation by the proteasome in a ubiquitin-independent manner. Using state-of-the-art cell-based HBV infection models, Cheng et al.17 showed that both NQO1 knockdown as well as treatment with dicoumarol decrease the levels of all HBV parameters except for cccDNA. The antiviral effect of dicoumarol was confirmed in vivo in 2 different mouse models. From a mechanistic point of view, Cheng et al.17 showed that overexpression of NQO1, and the subsequent increase in the levels of HBx and its association with cccDNA, leads to increased HBV RNA transcription from cccDNA. Reciprocally, inhibition of NQO1 (by genetic or chemical approaches) decreases the association of HBx with cccDNA, increases repressive marks on cccDNA-associated histones and decreases the levels of HBV RNAs both in vitro and in vivo. Finally, Cheng et al. observed a partial rescue of the levels Smc5/Smc6 proteins in cells transfected with a vector expressing HBx and treated with dicoumarol. Why this was not observed in HBV-infected cells remains to be determined. Of note, the higher expression of NQO1 in HBV permissive cancer cell lines, such as HepG2, compared to normal hepatocytes19 may explain some of the discrepancies observed regarding the role of HBx and the intensity of HBV transcription/replication in different cell types.
According to Cheng et al.,17 the antiviral effect of dicoumarol seemed to be mainly due to its activity against NQO1, since genetic perturbation of NQO1 levels leads to drug-equivalent phenotypes against HBV. NQO1 is a protein with multiple protectives roles including some that extend beyond its catalytic function as a quinone reductase. Indeed, besides its antioxidant functions, NQO1 was shown to bind to selective proteins such as p53 and prevent their degradation by the proteasome (for review see20
). The data from Cheng et al.17 suggest that HBx benefits from this NQO1 “gatekeeper” role. Unfortunately, the detailed mechanisms explaining NQO1 selectivity regarding the protection of given proteins have not yet been elucidated. Collectively, increasing HBx degradation by decreasing NQO1 activity is an interesting option to block HBV transcription and thereby the production of HBV proteins, as well as to block HBx-induced host dysregulation that can lead to carcinogenesis.8 However, it should be considered that reduced NQO1 activity has been associated with a higher risk of cancer development.21 NQO1 activity was also shown to be increased in some forms of cancer, including liver cancer,19
and blockage of NQO1 by dicoumarol inhibits cell growth and seeding efficiency in soft agar of pancreatic cancer cells.22
In this regard, efforts have been made to identify other molecules that selectively inhibit NQO1 (for review see23 ), however the clinical translation of this approach for cancer prevention or treatment remains to be determined.
Beside its activity against NQO1, dicoumarol has several other pleiotropic effects. Originally dicoumarol was uncovered as a natural anticoagulant (based on its chemical similarity to vitamin K) and was in clinical use for decades as an oral drug to prevent thrombogenesis.18 Interestingly, dicoumarol also has been described to exert antimicrobial and antiviral activities.18
However, safety issues such as hemorrhage, anemia, neurotoxicity or periodontal toxicity18 have resulted in replacement of dicoumarol with next generation vitamin K antagonists. Even though Cheng et al.17 estimated that the human equivalent dose of dicoumarol required for anti-HBV properties may be in a range for therapeutic use as an anticoagulant, it remains to be determined if the drug would fulfil modern safety requirements for long-term treatment. Moreover, since dicoumarol targets the HBx protein and not the cccDNA itself, withdrawal of the treatment would likely lead to rebound of viral transcription and replication.
In summary, Cheng et al.17 provided new insights into the regulation of HBx by cellular factors as well as the proof of concept that targeting HBx by chemical compounds is a valuable antiviral strategy. While the clinical application of dicoumarol may be challenging in this setting, because of its safety profile, the identification of new molecules specifically targeting the interaction between HBx and NQO1, without fully inhibiting the physiological activities of NQO1, may provide a new approach to enlarge our armamentarium against HBV.

StephenW

通过降解HBx蛋白来沉默HBV附加体：走向功能性治疗？

    朱莉·卢西福拉（Julie Lucifora）
    托马斯·鲍默特

发布时间：2020年11月10日DOI：https：//doi.org/10.1016/j.jhep.2020.10.018
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参见文章，第522–534页
慢性HBV感染是全世界晚期肝病和癌症的主要原因，对全球健康构成了挑战。尽管批准的疗法可以有效地控制HBV病毒血症，但很少能消除病毒。 HBV的生命周期显示出一些独特的功能，使病毒能够在肝细胞中持续存在。通过仅在新形成的核衣壳中复制其基因组，HBV逃避了细胞抗病毒免疫反应的识别。而且，HBV还在感染细胞的核内建立稳定的附加体。这种附加体，称为cccDNA（共价闭合的环状DNA），也被称为“微型染色体”。除了没有复制起点外，HBV cccDNA分子确实组织成染色质样结构，在电子显微镜下显示出典型的串珠状排列。1HBV cccDNA充当了所有转录的模板HBV RNA由细胞RNA聚合酶II产生。对其转录活性的调节导致组蛋白，组蛋白修饰酶和几种细胞转录因子的募集。2、3、4、5、6。尽管长期争论不休，但病毒蛋白HBx在控制HBV转录中的作用逐渐成为现实在过去的十年中更加清晰（回顾参见7,8）。总的来说，几项不同的独立研究表明，HBx是cccDNA驱动的HBV RNA转录的起始和维持所必需的。6,9,10,11HBx最有可能通过不同和互补的机制发挥这一关键作用：HBx促进降解染色体复合物Smc5 / 6的结构维持的功能，最近被鉴定为抑制cccDNA RNA转录的宿主限制因子。12，13，14还显示了HBx直接募集到cccDNA中。 HBx的缺乏会导致cccDNA结合的组蛋白的乙酰化降低，转录共激活因子p300的募集受损，以及组蛋白脱乙酰基酶3和异染色质蛋白因子1（HP1）的募集。6此外，许多研究（尽管通常在生理过度表达较少的系统中进行） ）已经报道了HBx对宿主细胞通路（如凋亡，钙信号传导和细胞周期）的扰动的影响，这些通路与肝脏疾病和肝癌发生有关（综述参见8,15）。由于HBx在HBV感染中起着核心作用，并且很可能与病毒引起的肝病有关，因此它是治疗慢性HBV感染的新疗法的一个有趣目标。该概念与其他旨在“功能性治愈”的方法相一致，其特征是持续丢失HBsAg（有或没有抗HBs血清转化），并改善了临床疗效。16为了鉴定靶向HBx的化合物，Cheng等人[17]筛选了小分子文库抑制HBx表达的能力。他们使用这种方法鉴定了双香豆酚，它可以剂量依赖性地降低HBx蛋白的表达，而不影响HBx mRNA的水平。在多种特性中，双香豆酚是NAD（P）H：醌氧化还原酶（NQO1）的竞争性抑制剂。18丧失功能和获得功能的测定表明NQO1与HBx结合并通过防止其被蛋白酶体降解而增强其稳定性。独立于泛素的方式。 Cheng等人[17]使用基于细胞的最新HBV感染模型，发现NQO1敲除以及用双香豆酚治疗均会降低除cccDNA以外的所有HBV参数水平。在两种不同的小鼠模型中体内证实了双香豆酚的抗病毒作用。从机理的角度看，Cheng等人[17]表明，NQO1的过表达，以及随后HBx水平的增加及其与cccDNA的关联，导致HBV RNA从cccDNA转录的增加。相应地，抑制NQO1（通过遗传或化学方法）可降低HBx与cccDNA的结合，增加cccDNA相关组蛋白的阻遏标记，并降低体内和体外HBV RNA的水平。最后，Cheng等。观察到部分拯救了表达HBx的载体转染并用双香豆酚处理过的细胞中Smc5 / Smc6蛋白的水平。为什么在HBV感染的细胞中未观察到这一点，尚待确定。值得注意的是，与正常肝细胞相比，NQO1在HBV允许的癌细胞系（例如HepG2）中的表达更高19可能解释了观察到的关于HBx的作用和HBV在不同细胞类型中转录/复制强度的差异。
根据Cheng等人的研究[17]，双香豆酚的抗病毒作用似乎主要是由于其对NQO1的活性，因为NQO1水平的遗传扰动导致了抗HBV的药物等效表型。 NQO1是一种具有多种保护作用的蛋白质，其中一些作用超出了其作为醌还原酶的催化功能。的确，除了其抗氧化功能外，NQO1还可以与诸如p53等选择性蛋白质结合，并防止其被蛋白酶体降解（综述见20）。
）。 Cheng等人[17]的数据表明，HBx受益于NQO1的“网守”角色。不幸的是，尚未阐明解释有关给定蛋白质保护的NQO1选择性的详细机制。总的来说，通过降低NQO1活性来增加HBx降解是阻止HBV转录从而阻止HBV蛋白产生以及阻止HBx诱导的可能导致癌变的宿主失调的一个有趣的选择。8但是，应考虑降低NQO1活性与罹患癌症的风险较高相关。21NQO1活性在某些形式的癌症（包括肝癌）中也表现出增加19
和双香豆酚阻滞NQO1抑制胰腺癌细胞软琼脂中的细胞生长和接种效率。22
在这方面，已经做出努力以鉴定选择性抑制NQO1的其他分子（综述见23），但是该方法用于癌症预防或治疗的临床翻译仍有待确定。
除其对NQO1的活性外，双香豆酚还具有其他多种功效。最初发现双香豆酚是一种天然抗凝剂（基于其与维生素K的化学相似性），并且已在临床上用作预防血栓形成的口服药物数十年。18有趣的是，双香豆酚还被描述具有抗微生物和抗病毒活性。18
但是，诸如出血，贫血，神经毒性或牙周毒性等安全问题已导致用下一代维生素K拮抗剂替代地香豆酚。即使Cheng等人[17]估计抗乙肝病毒特性所需的人等效剂量的双香豆酚可能在治疗用途中作为抗凝剂，但仍需确定该药物能否长期满足现代安全性要求治疗。此外，由于双香豆酚靶向HBx蛋白而不是cccDNA本身，因此退出治疗可能会导致病毒转录和复制反弹。
综上所述，Cheng等人[17]为细胞因子对HBx的调控提供了新的见识，并提出了化学化合物靶向HBx是一种有价值的抗病毒策略的概念证明。尽管在这种情况下双香豆酚的临床应用可能会面临挑战，但由于其安全性，在不完全抑制NQO1的生理活性的情况下，鉴定专门针对HBx和NQO1相互作用的新分子，可能会提供一种新的方法来扩大我们的乙肝疫苗库。