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/
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
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
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.
