Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA.
Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA.
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DOI：
10.1371/journal.ppat.1010576
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发表时间：
2022-06
期刊：
影响因子：
6.7
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中科院分区：
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B型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）作为HBV感染的病毒持久形式和转录模板，劫持宿主组蛋白和非组蛋白蛋白形成微型染色体，并利用翻译后修饰（PTM）“组蛋白密码”进行转录调控。HBV X蛋白（HBx）被称为cccDNA转录激活因子。在这项研究中，我们建立了一个诱导型报告细胞系的双重系统，模拟野生型（wt）和HBx无效HBV的感染，两者都分泌HA标记的HBeAg作为cccDNA转录的半定量标志物。使用染色质免疫沉淀结合定量PCR（ChIP-qPCR）对wt和HBx无效系统的cccDNA结合组蛋白PTM分析证实，HBx对于维持cccDNA处于转录活性状态至关重要，其特征在于活性组蛋白PTM标记物。在wt和HBx无效HBV细胞系中建立的cccDNA微型染色体的差异蛋白质组学分析揭示了组特异性命中。在HBx缺陷条件下的命中之一是非组蛋白宿主DNA结合蛋白高迁移率族蛋白1（HMGB 1）。在HBV稳定细胞系和体外感染系统中，通过ChIP-qPCR试验验证了其与HBx无效cccDNA的升高相关性。此外，在HBx无效HBV诱导型和感染模型中HMGB 1的实验性下调导致cccDNA微型染色体的转录再激活，伴随着cccDNA相关组蛋白转换为常染色质状态，激活组蛋白PTM景观和随后的cccDNA转录上调。在机制上，HBx与HMGB 1相互作用并阻止其与cccDNA结合，而不影响HMGB 1的稳态水平。综上所述，我们的研究结果表明，HMGB 1是HBV cccDNA的一种新的宿主限制因子，具有表观遗传沉默机制，可以被病毒转录激活因子HBx抵消。目前，B型肝炎病毒（HBV）的流行仍然是全球医疗保健的重大负担。由于缺乏能够消除HBV DNA基因组的肝内持久形式（即共价闭合环状DNA（cccDNA））的疗法，迄今为止还没有治愈慢性HBV感染的方法。在受感染的肝细胞内，cccDNA劫持宿主结构和调节因子以组装成微型染色体，但某些内在宿主因子能够抵消cccDNA活性。在我们的研究中，我们鉴定了一种抗HBV宿主限制因子，特别是高迁移率族蛋白1（HMGB 1），并证明了其介导cccDNA的表观遗传沉默（即功能失活）的潜力。然而，病毒编码的辅助蛋白HBx能够拮抗HMGB 1并维持cccDNA的活性状态。这可能有助于更好地理解HBV感染期间病毒-宿主相互作用，以及HBV感染表观遗传药物的开发和重新考虑癌症治疗策略，其中HMGB 1被用作抗癌靶点。
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) “histone code” for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx. Nowadays hepatitis B virus (HBV) prevalence remains a significant burden to the worldwide healthcare. There is no cure for chronic HBV infection so far due to the lack of therapies that enable elimination of an intrahepatic persistent form of HBV DNA genome, namely, the covalently closed circular DNA (cccDNA). Inside the infected liver cells, cccDNA hijacks host structural and regulatory factors to assemble into a minichromosome, but certain intrinsic host factors are able to counteract cccDNA activity. In our study, we identified an anti-HBV host restriction factor, specifically the high mobility group box 1 protein (HMGB1), and demonstrated its potential to mediate an epigenetic silencing, i.e. functional inactivation, of the cccDNA. However, the virally encoded accessory protein HBx is able to antagonize HMGB1 and maintain an active state of cccDNA. This may contribute to a better understanding of virus-host interaction during HBV infection, and to the development of HBV infection epigenetic drugs and re-consideration of cancer therapeutics strategies, where HMGB1 is used as an anti-cancer target.
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DOI：
10.3390/v9030056
发表时间：
2017-03-21
期刊：
Viruses
影响因子：
--
作者：
Hu J;Liu K
通讯作者：
Liu K
影响因子：
30.8
作者：
Calogero, S;Grassi, F;Bianchi, ME
通讯作者：
Bianchi, ME
DOI：
10.1007/978-1-62703-484-5_13
发表时间：
2013
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Cai, Dawei;Nie, Hui;Yan, Ran;Guo, Ju-Tao;Block, Timothy M;Guo, Haitao
通讯作者：
Guo, Haitao
影响因子：
7.6
作者：
Cai, Dawei;Wang, Xiaohe;Yan, Ran;Mao, Richeng;Liu, Yuanjie;Ji, Changhua;Cuconati, Andrea;Guo, Haitao
通讯作者：
Guo, Haitao
影响因子：
5.3
作者：
El Gazzar, Mohamed;Yoza, Barbara K.;McCall, Charles E.
通讯作者：
McCall, Charles E.