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标题: HBV cccDNA的:病毒持续感染水库主要障碍慢性乙型肝炎的治疗 [打印本页]

作者: StephenW    时间: 2015-6-7 15:39     标题: HBV cccDNA的:病毒持续感染水库主要障碍慢性乙型肝炎的治疗

Gut doi:10.1136/gutjnl-2015-309809

    Recent advances in basic science

HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B
Open Access

    Michael Nassal

    Correspondence to Dr Michael Nassal, Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Hugstetter Str. 55, Freiburg D-79106, Germany; [email protected]

    Received 17 April 2015
    Revised 12 May 2015
    Accepted 13 May 2015
    Published Online First 5 June 2015

Abstract

At least 250 million people worldwide are chronically infected with HBV, a small hepatotropic DNA virus that replicates through reverse transcription. Chronic infection greatly increases the risk for terminal liver disease. Current therapies rarely achieve a cure due to the refractory nature of an intracellular viral replication intermediate termed covalently closed circular (ccc) DNA. Upon infection, cccDNA is generated as a plasmid-like episome in the host cell nucleus from the protein-linked relaxed circular (RC) DNA genome in incoming virions. Its fundamental role is that as template for all viral RNAs, and in consequence new virions. Biosynthesis of RC-DNA by reverse transcription of the viral pregenomic RNA is now understood in considerable detail, yet conversion of RC-DNA to cccDNA is still obscure, foremostly due to the lack of feasible, cccDNA-dependent assay systems. Conceptual and recent experimental data link cccDNA formation to cellular DNA repair, which is increasingly appreciated as a critical interface between cells and viruses. Together with new in vitro HBV infection systems, based on the identification of the bile acid transporter sodium taurocholate cotransporting polypeptide as an HBV entry receptor, this offers novel opportunities to decipher, and eventually interfere with, formation of the HBV persistence reservoir. After a brief overview of the role of cccDNA in the HBV infectious cycle, this review aims to summarise current knowledge on cccDNA molecular biology, to highlight the experimental restrictions that have hitherto hampered faster progress and to discuss cccDNA as target for new, potentially curative therapies of chronic hepatitis B.

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/



作者: StephenW    时间: 2015-6-7 15:40

肠道DOI:10.1136 / gutjnl-2015-309809

    在基础科学的最新进展

HBV cccDNA的:病毒持续感染水库主要障碍慢性乙型肝炎的治疗
开放存取

    迈克尔Nassal

    函授博士迈克尔Nassal,内科二科/分子生物学,大学医​​院弗赖堡,Hugstetter海峡。 55,弗赖堡D-79106,德国; [email protected]

    收到2015年4月17日
    修订后的2015年5月12日
    接受2015年5月13日
    网上公布的第一2015年6月5日

抽象

至少有2.5亿人的全球慢性感染乙肝病毒,小嗜肝DNA病毒,通过复制反转录。慢性感染大大增加了终端肝脏疾病的风险。目前的治疗方法很少能达到治愈,由于细胞内的病毒复制中间被称为共价闭合环状(CCC)DNA的耐火性质。一旦感染,cccDNA的产生是从蛋白偶联轻松圆(RC)的DNA病毒粒子进入的基因组中的宿主细胞细胞核的质粒般的游离体。它的基本作用是作为模板的所有病毒RNA,并在后果新的病毒体。的RC-DNA的病毒前基因组RNA的逆转录生物合成现在被理解相当详细,但转化率的RC-DNA与cccDNA的仍然是模糊的,foremostly由于缺乏可行,cccDNA的依赖性测定系统。概念和最近的实验数据链路cccDNA的形成,细胞DNA修复,这是越来越多赞赏,因为细胞和病毒之间的重要接口。连同新的体外HBV感染系统的基础上,胆汁酸转运牛磺胆酸钠cotransporting多肽作为HBV条目受体的鉴定,这提供了新颖的机会破译,并最终干扰,在形成的HBV持久储存器。的cccDNA在乙肝病毒感染周期中的作用的简要介绍后,本次审查的目的是总结现有的知识对cccDNA的分子生物学,突出实验的限制,迄今阻碍了更快的进展,并讨论cccDNA的目标为新的,可能治愈疗法慢性乙型肝炎

这是分布在依照知识共享署名非商业(CC BY-NC 4.0)牌照,允许他人分发,混音,改编,建立在此工作的非商业化,并授权他们在不同的衍生作品的开放存取的文章来说,所提供的原始工作正确的引用和使用是非商业性。请参阅:http://creativecommons.org/licenses/by-nc/4.0/
作者: StephenW    时间: 2015-6-7 15:46

Ridding the liver of cccDNA

Steady-state cccDNA levels are determined by the rates of formation versus loss. As long as the turnover kinetics are not settled, active elimination of existing cccDNA appears as the most straightforward approach. Two major current strategies are to mimic, in the chronic setting, the immune-mediated clearance of most of the cccDNA that occurs during self-limited acute HBV infection13 ,132 (figure 7D) and the employment of designer nucleases that have revolutionised genome editing (figure 7E).

Innate and adaptive immune responses are critical in clearing acute HBV infection. Restoring full activity of the insufficient immune responses typical for chronic HBV infection will thus remain highly relevant,12 likely on both the cellular level (to clear infected hepatocytes) and the humoral level (to prevent reinfection). Given the breadth of the field, only a few general and simplified considerations are discussed here (for more comprehensive accounts, see references14 ,15 ,133 ,134).

The two extreme scenarios for cccDNA clearance from hepatocytes are non-cytolytic elimination (‘curing’), or destruction of (nearly) all cells harbouring cccDNA by T cells (‘killing’) and replacement by non-infected cells.132 Frequent liver damage during chronic hepatitis B argues against curing as the only explanation; conversely, the fast recovery from acute infection would require that the entire liver be turned over within a few weeks—while maintaining functionality. Hence, likely both mechanisms exist, yet their relative contributions are still debated, owing to the multiple, difficult to assess parameters involved.90 ,91 ,135 Examples include the fate of cccDNA during cell division,136 specifically if and how cccDNA re-enters the reforming nucleus; the turnover time of cccDNA-free versus cccDNA bearing cells and, for the latter, the impact of cccDNA transcriptional activity; or the origin, fraction and proliferation characteristics of cells that are refractory to infection, or refractory to immune-mediated clearance. Irrespective of these difficulties, cytokines such as interferons and their downstream effectors appear to play an important role, although the exact mechanisms are not firmly established. While various steps of the replication cycle might be affected,132 a recent study137 suggested that very-high-dose interferon-α, or more potently activation of the lymphotoxin-β receptor, could directly target cccDNA integrity via APOBEC3A and 3B-mediated deamination of the (-)-strand and subsequent degradation. Though some aspects are controversial,138 ,139 the worthiness of activating innate responses is underlined by promising preclinical results with the Toll-like receptor 7 agonist GS-9620.140

Notably, in various settings of immune-mediated cccDNA decline, a fraction of the cccDNA pool appeared refractory to further reduction.88 ,84 ,131 ,137 ,141 This might reflect properties of the cccDNA harbouring cell, or cccDNA may per se exist in distinct forms that differ, for example, in methylation, chromatinisation or some unknown property (figure 3). Possibly, non-natural ways to induce cccDNA degradation might be able to also target this resilient reservoir.
Box 1
Key role of HBV covalently closed circular (ccc)DNA in viral persistence and chronic hepatitis B

Chronic hepatitis B, caused by persistent infection with HBV, puts >250 million people at risk to develop terminal liver disease.

HBV persistence is mediated by an intranuclear, episomal form of the viral genome called cccDNA.

cccDNA is the template for viral RNAs and subsequent generation of progeny virions.

A few copies of cccDNA per liver can (re)initiate full-blown infection.

cccDNA is not targeted by current treatments—but a cure of chronic hepatitis B will require elimination of cccDNA.

Recent advances, including identification of a liver-specific HBV receptor and evidence for HBV's interaction with cellular DNA damage repair, promise to greatly expand the limited knowledge on cccDNA biology.
Box 2
Major unresolved issues in HBV covalently closed circular (ccc)DNA biology

Does the longevity of cccDNA relate to individual molecules, or is there cccDNA turnover? If turnover occurs, by which route (intracellular? cell-to-cell? with an extracellular phase?) and with which kinetics?

How is cccDNA ‘cleared’ in acute self-limiting hepatitis B?

If cells can be immunologically cured from cccDNA, by which mechanism(s)?

Does cccDNA survive cell division—and how?

What restricts HBV cccDNA formation/accumulation in most human hepatoma cell lines and particularly in mouse hepatocytes?

Why is this restriction much less pronounced for duck HBV, even in human cells?

Which mechanism(s) prevent infinite cccDNA amplification even in duck HBV model systems? Could such mechanisms be harnessed to reduce cccDNA copy number?

Advances in genome editing using designer nucleases115 have prompted studies harnessing these new tools for targeting cccDNA, for example, by zinc-finger nucleases,142 ,143 transcription activator-like endonucleases144 or the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.85 However, numerous issues are unresolved, foremost efficient access of the nucleases to all cccDNA molecules. Unless cccDNA-bearing cells can specifically be targeted, the nucleases must be delivered to all hepatocytes. Off-target effects, including chromosomal integration of the linearised viral DNA, could adversely affect hepatocyte function, especially when long-term presence of the effector nucleases is necessary. Also, while NHEJ-mediated repair of the nuclease-induced DSBs is error-prone,47 a fraction of repair events will result in the reformation of intact cccDNA. Not the least, it is unclear how the excess RC-DNA in the same cells affects the targeting efficiency for cccDNA. Again, much more research is required and there is ample room for other strategies including therapeutic vaccination or anti-sense and RNA-interference-based approaches.54
Conclusions and perspectives

The importance of cccDNA as persistence reservoir of HBV is firmly established and so is the realisation that any strategy towards a cure of chronic hepatitis B will have to cope with this long-lived molecule. Current knowledge on cccDNA formation and degradation is still very limited, yet in particular the emerging cell culture infection systems, and the possible development of small animal infection models, promise to dramatically change this situation. Still, the current gap in knowledge is so large that many facets of cccDNA biology are open to new discoveries (box 2). It appears unlikely that a single magic bullet will turn up that causes cccDNA to completely disappear; however, the combination of new knowledge on cccDNA biochemistry, a molecular understanding of how the body's immune system deals with cccDNA during clearance of acute HBV infection, and new technologies for targeted DNA manipulation hold promise to achieve this goal. An indispensable premise is appropriate funding for basic HBV research. New opportunities might come from the renewed interest of pharmaceutical industry in HBV. It is hoped that fair and as far as possible open interactions between academia and industry will make the quest for a cure of chronic hepatitis B a similar success as in the case of chronic hepatitis C.145
Acknowledgments

I apologise to numerous colleagues whose original contributions could not or only partly be referenced for space limitations.
Footnotes

    Funding Work in the author's laboratory was supported by the Deutsche Forschungsgemeinschaft (DFG) via grant NA154/12-2 within the Collaborative Research Unit FOR1202 (persistence of hepatotropic viruses) and by the European Union via the FP7 Infect-ERA programme (project ID hepBccc).

    Competing interests None declared.

    Provenance and peer review Commissioned; externally peer reviewed.

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

作者: StephenW    时间: 2015-6-7 15:47

摆脱的cccDNA的肝

稳态水平的cccDNA通过形成对比损失的比率确定。只要营业额动力学没有谈妥,积极消除现存的cccDNA似乎是最直接的方法。两大当前策略是模仿,在慢性设置,大多数在自限性急性HBV infection13发生的cccDNA的,132(图7D),并且已经彻底改变基因组编辑设计师核酸酶的就业免疫介导的清除(图7E)。

先天和适应性免疫反应是在清除急性HBV感染的关键。恢复典型的慢性HBV感染的免疫反应不够充分的活动将因此保持高度相关性,12可能同时在细胞水平(以清除感染的肝细胞)和体液免疫水平(以防止再感染)。给定字段的广度,只有少数一般和简化的考虑这里讨论(对于更为全面,参照下述references14,15,133,134)。

两种极端场景的cccDNA间隙从肝细胞是非溶细胞消除(“固化”),或破坏(几乎)所有细胞通过T细胞窝藏的cccDNA的('杀')和更换由非感染cells.132频繁肝损伤慢性乙型肝炎时反驳了固化作为唯一的解释;相反,快速恢复从急性感染,需要整个肝脏可以在几个星期内,同时维持功能性翻转。因此,很可能这两种机制的存在,但它们的相对贡献仍有争议,由于多个,难以评估参数involved.90,91,135的实例包括细胞分裂期间的cccDNA的命运,136具体是否以及如何cccDNA的重新进入重整核;的周转时间的cccDNA - 自由与cccDNA的轴承细胞和,对于后者,cccDNA的转录活性的影响;或细胞的起源,分数和增殖特性是难治的感染,或难治性免疫介导的清除。不论这些困难,细胞因子如干扰素和其下游效应似乎发挥着重要作用,但确切的机理尚未确立。而可能受到影响的各种步骤的复制周期,132最近study137表明非常高的剂量的α干扰素,或更有力地活化的淋巴毒素β受体,可以直接靶向经由APOBEC3A cccDNA的完整性和3B介导的脱氨的( - ) - 链和随后的降解。虽然有些方面是有争议的,138,激活固有响应139的老有所为被看好临床前结果与Toll样受体激动剂7 GS-9620.140下划线

值得注意的是,在免疫介导的cccDNA下降各种设置,所述的cccDNA池的一小部分出现耐火进一步reduction.88,84,131,137,141,这可能反映了cccDNA的性质窝藏细胞,或cccDNA的可能本身存在于不同的形式而不同,例如,在甲基化,chromatinisation或某种未知的(图3)。也许,不自然的方式来诱导的cccDNA降解也许能也瞄准这一弹性水库。
专栏1
乙肝病毒的主要作用在病毒持续感染和慢性乙型肝炎共价闭合环状(CCC)的DNA

慢性乙型肝炎,乙肝病毒引起的持续性感染,使> 2.5亿人处于危险之中,开发终末期肝病。

乙肝病毒的持久性是由核内,病毒基因组被称为的cccDNA的附加体形式介导的。

cccDNA的对病毒RNA和后续世代后代病毒体的模板。

每肝脏可以(重新)cccDNA的几份开始全面爆发感染。

cccDNA的不是由目前的治疗,但是慢性乙型肝炎的治疗,需要消除的cccDNA针对性。

最新进展,其中包括鉴定肝特异性受体HBV和证据为HBV的与细胞DNA损伤修复的互动,承诺极大地扩展cccDNA的生物学知识有限。
专栏2
乙肝病毒主要的未解决的问题共价闭合环状(CCC)的DNA生物

是否cccDNA的长寿涉及到单个分子,或者是有cccDNA的成交?如果发生变化,通过该途径(胞内β细胞 - 细胞?有细胞外相位α)和与动力学周转?

如何cccDNA的'清除'的急性自限性乙肝?

如果细胞可以从免疫治愈的cc​​cDNA,其中机制(S)?

难道cccDNA的生存细胞的分裂,以及如何?

是什么限制了HBV cccDNA的形成/堆积在大多数人肝癌细胞系,特别是在小鼠肝细胞?

为什么这种限制要少得多宣判鸭乙型肝炎病毒,即使在人体细胞?

哪种机制(S)防止无限放大的cccDNA即使在鸭乙肝模型系统?难道这样的机制加以利用,以减少cccDNA的拷贝数?

进展在基因组中使用设计师nucleases115已促使研究利用这些新的工具用于靶向cccDNA的,例如,通过锌指核酸酶,142,143转录激活状endonucleases144或RNA的引导簇定期相互间隔短回文重复序列(CRISPR)编辑/ CAS system.85然而,许多问题都是核酸对所有的cccDNA分子未解决,最重要的是有效的访问。除非cccDNA的轴承单元可以特异性靶向的核酸必须被传递到所有肝细胞。脱靶效应,其中包括线性化病毒DNA的染色体整合,可以肝功能产生不利的影响,特别是当效应子核酸的长期存在是必要的。此外,虽然核酸酶诱导的DNA双链断裂NHEJ介导的修复是容易出错,维修事件47的一小部分会导致cccDNA的完整的改革。并非最不重要的,目前还不清楚过量RC-DNA在同一细胞如何会影响cccDNA的靶向效率。此外,更多的研究是必要的,有足够的空间用于其它战略,包括治疗性疫苗接种或反义和RNA干扰为基础的approaches.54
结论和观点

的cccDNA作为乙肝病毒的持久性水库的重要性,牢固确立,因此是实现了对慢性乙肝的治疗任何战略必须应对这种长寿的分子。对cccDNA的形成和恶化现有的知识还十分有限,但特别是新兴的细胞培养系统的感染,以及小动物感染模型的可能发展,承诺极大地改变这种状况。不过,目前的差距在知识是如此之大,cccDNA的生物学的许多方面是开放的新发现(盒2)。这似乎不太可能,一个神奇的子弹就会转起来,导致cccDNA的完全消失;然而,对cccDNA的生物化学,人体的免疫系统处理的cccDNA的急性HBV感染的间隙中,并进行有针对性的DNA操作大有前途的新技术是如何实现这一目标的分子理解新知识的结合。一个不可缺少的前提,是基础研究HBV适当的资金。新的机会可能来自制药业的HBV重新产生了兴趣。人们希望,公正,尽可能学术界和工业界之间可能存在的相互作用开放将使寻求慢性乙型肝炎的治疗类似的成功,因为在慢性肝炎C.145的情况下
致谢

我道歉,许多同事的原创性贡献无法或只能部分地空间的限制引用。
脚注

    在笔者的实验室工作经费由德意志研究联合会(DFG),通过资助NA154 / 12-2内的合作研究单位FOR1202(嗜肝病毒的持久性)和欧盟通过FP7传染-ERA计划(项目编号hepBccc支持)。

    相互竞争的利益无申报。

    出处和同行评审委托;外部同行审查。

这是分布在依照知识共享署名非商业(CC BY-NC 4.0)牌照,允许他人分发,混音,改编,建立在此工作的非商业化,并授权他们在不同的衍生作品的开放存取的文章来说,所提供的原始工作正确的引用和使用是非商业性。请参阅:http://creativecommons.org/licenses/by-nc/4.0/
作者: StephenW    时间: 2015-6-7 15:48

整篇文章:
http://gut.bmj.com/content/early ... nl-2015-309809.full
作者: 君看一叶舟    时间: 2015-6-7 19:52

此文创新之处是哪里?
作者: StephenW    时间: 2015-6-7 20:00

君看一叶舟 发表于 2015-6-7 19:52
此文创新之处是哪里?

this review aims to summarise current knowledge on cccDNA molecular biology, to highlight the experimental restrictions that have hitherto hampered faster progress and to discuss cccDNA as target for new, potentially curative therapies of chronic hepatitis B.

没有创新之处.




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