乙肝难治的原因-共价闭合环状DNA(cccDNA)
HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B介绍
全世界至少有2.5亿人慢性感染了HBV1病毒，并大大增加了发生肝纤维化、肝硬化和肝细胞癌的风险，每年估计造成65万人死亡。虽然有一种有效的预防疫苗可用，但目前治疗慢性乙型肝炎的3种方法仅限于1型干扰素和5种核苷酸类似物(NAs)，它们的目标是病毒聚合酶，P蛋白，一种多功能的逆转录酶(见下文)。由于严重的副作用，只有一小部分患者适合接受干扰素治疗，而少于10%的患者表现出持续的病毒学反应，以乙型肝炎表面抗原(HBsAg)的损失来衡量;见下文)。4个NAs耐受性好得多，最有效的药物恩替卡韦和替诺福韦可以将病毒血症降低5-6个log，通常低于检测限，病毒耐药率低。然而，HBsAg清除非常罕见(0-5%)，即使在长期治疗后，停药后频繁的病毒反弹，表明需要终身治疗。在免疫抑制的情况下，几十年前急性乙肝病毒感染的患者甚至可以在免疫抑制后重新激活，表明病毒可以免疫控制但不能消除。
这种持久性的病毒学关键是细胞内的HBV复制中间体，称为共价闭合环状DNA(cccDNA) ，它驻留在受感染细胞的细胞核中，作为一种外体(即非整合)质粒样分子，产生子代病毒。因此，慢性乙型肝炎的治疗需要消除cccDNA。然而，尽管研究了超过30年，关于cccDNA形成和降解的分子机制仍知之甚少，主要原因是缺乏合适的实验系统。最近的发现将改变这一状况，特别是肝细胞内胆汁酸转运体，牛胆酸钠共转运多肽(NTCP)的鉴定;也被称为SLC10A1)，是HBV和丁型肝炎病毒(HDV)的进入受体，为HBV进入干细胞打开了大门(盒子1)。10、11
HBV持续存在的第二个关键是有缺陷的免疫反应，通常包括功能衰竭和细胞毒性T细胞的耗尽，缺乏足够的CD4+ T细胞的帮助，以及未能装配中和抗体。虽然即使找到了其他减少cccDNA的方法，免疫恢复也可能是不可缺少的，但要获得更多信息，请参考相关评论。13-16本文的重点将是cccDNA研究及其实验困难的简史，以及最近的进展，以及如何将其转化为治疗慢性乙型肝炎的新疗法。
Figure 7
靶向HBV共价闭合环(ccc)DNA的候选治疗策略。在宿主细胞核中，圆形代表P蛋白连接，无蛋白松弛圆形(RC)-DNA和cccDNA。(A)通过阻断参与RC-DNA到cccDNA转换的多个步骤的宿主因子，预防cccDNA积累。(B)预防以衣壳为靶点的药物核输入RC-DNA;此外，RC-DNA的胞质释放可能触发DNA传感器，如循环GMP-AMP合成酶(cGAS)，并激活STING，诱导抗病毒细胞因子。(C)通过诱导宿主细胞表观遗传机制或阻断HBV X蛋白(HBx)去沉默cccDNA转录活性。(D)通过免疫介导机制(可能通过APOBEC酶)降解现有cccDNA，或者(E)通过直接靶向设计核酸酶(用于基因组编辑)。详见文本。
Candidate therapeutic strategies to target HBV covalently closed circular (ccc)DNA. The circles represent P protein-linked and P protein-free relaxed circular (RC)-DNA and cccDNA in the host cell nucleus. (A) Prevention of cccDNA accumulation by blocking host factors involved in the multiple steps of RC-DNA to cccDNA conversion. (B) Prevention of nuclear import of RC-DNA by capsid-targeting drugs; cytoplasmic release of RC-DNA may, in addition, trigger DNA sensors like cyclic GMP-AMP synthase (cGAS) and activate STING to induce antiviral cytokines. (C) Silencing of cccDNA transcriptional activity by inducing the host cell’s epigenetic machinery, or by blocking the de-silencing activity of HBV X protein (HBx). (D) Degradation of existing cccDNA by immune-mediated mechanisms, perhaps via APOBEC enzymes, or (E) by direct targeting with designer nucleases as used in genome editing. See text for details.
HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B
Introduction
At least 250 million people worldwide are chronically infected with HBV1 and at a greatly increased risk to develop liver fibrosis, cirrhosis and hepatocellular carcinoma, causing an estimated 650 000 deaths per year.2 While an efficient prophylactic vaccine is available,3 current treatments for chronic hepatitis B are limited to type 1 interferons and five approved nucleos(t)ide analogues (NAs), which target the viral polymerase, P protein, a multifunctional reverse transcriptase (see below). Due to severe side effects, only a fraction of patients are eligible for interferon therapy, and <10% of them show a sustained virological response, measured as loss of hepatitis B surface antigen (HBsAg; see below).4 NAs are much better tolerated, and the most potent drugs, entecavir and tenofovir, can reduce viraemia by 5–6 logs, often below detection limit, and with low rates of viral resistance development.5 However, HBsAg clearance is very rare (0–5%) even after prolonged treatment,4 and the frequent viral rebound upon therapy withdrawal indicates a need for lifelong treatment.6 Reactivation can even occur, upon immunosuppression, in patients who resolved an acute HBV infection decades ago,7 indicating that the virus can be immunologically controlled but is not eliminated.
The virological key to this persistence is an intracellular HBV replication intermediate, called covalently closed circular (ccc) DNA, which resides in the nucleus of infected cells as an episomal (ie, non-integrated) plasmid-like molecule that gives rise to progeny virus. A cure of chronic hepatitis B will therefore require elimination of cccDNA. However, despite >30 years of research, little is known about the molecular mechanisms of cccDNA formation and degradation, foremostly due to the lack of suitable experimental systems. Recent discoveries are about to change this situation, particularly the identification of a liver-resident bile acid transporter, sodium taurocholate cotransporting polypeptide (NTCP; also known as SLC10A1), as an entry receptor for HBV and hepatitis delta virus (HDV), which usurps HBV’s envelope to enter cells8 ,9 (box 1). Various aspects of this finding have recently been reviewed.10 ,11
A second key for HBV persistence is a flawed immune response, typically including the functional exhaustion and depletion of cytotoxic T cells, a lack of adequate CD4+ T cell help, and failure to mount neutralising antibodies. While immune restoration will likely be indispensable even if other ways are found to reduce cccDNA,12 for more information readers are referred to pertinent reviews.13–16 The focus here will be on a brief history on cccDNA research and its experimental difficulties, and on recent developments and how they may translate into new, curative treatments for chronic hepatitis B.keywords: CHRONIC VIRAL HEPATITIS; DNA DAMAGE; HEPATITIS B; MOLECULAR MECHANISMS
https://www.ncbi.nlm.nih.gov/pubmed/26048673
https://gut.bmj.com/content/64/12/1972
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