Molecular Mechanisms of HBV cccDNA Formation

HBV cccDNA形成的分子机制

• 批准号: 10656460
• 负责人: Haitao Guo
• 金额: $ 38.78万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-11 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656460
• 关键词: Adverse effects; Affect; Anabolism; Binding; Biochemistry; Biogenesis; Biology; Cell Line; Cell Nucleus; Cell physiology; Cells; Chemicals; Chronic; Chronic Hepatitis B; Circular DNA; Cirrhosis; Complement; Complex; Cytoplasm; DNA; DNA Ligases; DNA Repair; DNA Repair Gene; Data; Development; Drug resistance; Employment; Episome; Excision; Exhibits; Failure; Flap Endonucleases; Genes; Genetic Transcription; Genome; Genomics; Goals; Guanosine; HBV Animal Model; Hepatitis B Therapy; Hepatitis B Virus; Hepatocyte; Human; Immunocompetent; Individual; Infection; Interferon alpha; Life Cycle Stages; Ligation; Link; Liver; Longevity; Maintenance; Malignant neoplasm of liver; Maps; Mass Spectrum Analysis; Messenger RNA; Minor; Molecular; Molecular Biology; Mus; Mutagenesis; Nature; Nuclear; Nucleocapsid; Nucleotide Excision Repair; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Play; Polymerase; Predisposition; Primary carcinoma of the liver cells; Process; Production; Proteins; Proteome; Proteomics; Public Health; RNA; RNA primers; Relaxation; Reporting; Research; Reverse Transcription; Risk Factors; Role; Side; Signal Transduction; Study models; Technology; Transcript; Transportation; Viral; Viral Genome; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; Work; analog; chromatin immunoprecipitation; comparative; effective therapy; high risk; insight; knock-down; mouse model; novel; novel therapeutics; phosphodiester; phosphoric diester hydrolase; receptor; response; stable cell line; targeted treatment; terminal redundancy; timeline; transcriptome; tyrosyl-DNA phosphodiesterase; viral DNA; viral genomics; viral rebound

ABSTRACT Hepatitis B virus (HBV) covalently closed circular (ccc) DNA plays a central role in the establishment of viral infection and persistence, and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment with current approved medications. HBV cccDNA is established upon initial infection through conversion of the partially double stranded relaxed circular (rc) DNA viral genome containing terminal peculiarities, through employment of the host cell’s DNA repair mechanisms in the nucleus. The cccDNA episome levels are maintained through a replication cycle that involves retrotranscription of a cccDNA transcript, termed pregenomic RNA, into progeny rcDNA genomes, some of which are returned to the nucleus for conversion into cccDNA. The conversion of rcDNA into cccDNA requires the removal of a covalently-linked copy of the polymerase from the 5’ end of one of the DNA strands, and this “deproteination” step generates a DNA intermediate, the deproteinated rcDNA (DP-rcDNA), as precursor for cccDNA formation. The rcDNA deproteination is a trigger signal for transportation of HBV nucleocapsid containing mature viral DNA into nucleus, where the rcDNA to cccDNA conversion takes place. We have recently mapped the termini of cytoplasmic DP-rcDNA, which demonstrated that the viral polymerase and RNA primer are completely removed from rcDNA during deproteination, the plus strand DNA is further elongated but the terminal redundant sequence is maintained on DP-rcDNA. In addition, recent studies by us and others have identified a handful of host DNA repair factors involved in cccDNA formation. However, there are many molecular details yet to be elucidated for a better understanding of cccDNA biosynthesis, and the establishment of immunocompetent small animal model for HBV infection is hampered by the inability of cccDNA formation in mouse hepatocyte. In this research application, by making use of a battery of molecular biology, biochemistry, proteomics and genomics technologies, we propose to further elucidate the molecular mechanisms underlying the biogenesis of DP-rcDNA (Aim 1) and cccDNA (Aim 2), and to define the host determinant(s) for the failure of cccDNA formation in mouse hepatocyte (Aim 3). Our ultimate goal is to illustrate a coherent picture of the molecular mechanisms/pathway for HBV cccDNA formation. The accomplishment of this project will reveal new potential antiviral targets for treatment of hepatitis B and aid the development of a mouse model fully susceptible to HBV infection.

抽象的 丙型肝炎病毒（HBV）共价闭合（CCC）DNA在病毒的建立中起着核心作用 感染和持久性，是停止治疗后病毒反弹的基础， 即使在使用当前批准的药物进行扩展治疗后，可以治愈。 HBV CCCDNA是 通过转化部分双链松弛的圆形（RC）DNA在初始感染后建立 通过使用宿主细胞的DNA修复机制，含有终末特征的病毒基因组 在核中。 CCCDNA发作水平是通过复制周期维持的，涉及 CCCDNA转录本的retrotractracts，称为前基因组RNA，中后代rcDNA基因组，其中一些 将其返回到细胞核中以转换为CCCDNA。 rcDNA转换为cccDNA需要 从一个DNA链的5'末端取出聚合酶的共价链接副本，这 “剥夺”步骤会产生DNA中间体，DelepototeRcDNA（DP-RCDNA）作为前体 CCCDNA形成。 rcDNA剥夺是用于运输HBV Nucleocapsid的触发信号 含有成熟的病毒DNA到核中，在该核中进行rcDNA到CCCDNA转化。我们有 最近绘制了细胞质DP-RCDNA的末端，该末端证明了病毒聚合酶和RNA 在剥离过程中，底漆完全从rcDNA中取出，加链DNA进一步伸长，但 末端冗余序列保持在DP-RCDNA上。此外，我们和其他人最近的研究 已经确定了涉及CCCDNA形成的少数宿主DNA修复因子。但是，有很多 分子细节尚待阐明，以更好地了解CCCDNA生物合成，并 无法建立用于HBV感染的免疫能力的小动物模型。 小鼠肝细胞中的CCCDNA形成。在本研究应用中，使用一电位分子 生物学，生物化学，蛋白质组学和基因组学技术，我们建议进一步阐明分子 DP-RCDNA生物发生的基础机制（AIM 1）和CCCDNA（AIM 2），并定义宿主 确定性晶体中CCCDNA形成在小鼠肝细胞中的失败（AIM 3）。我们的最终目标是 图解了HBV CCCDNA形成的分子机制/途径的相干图片。这 该项目的实现将揭示用于治疗丙型肝炎的新潜在抗病毒靶标的 开发小鼠模型完全容易受到HBV感染的影响。

项目成果

Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B.

• DOI: 10.1016/j.antiviral.2018.08.014
• 发表时间: 2018-10
• 期刊: Antiviral research
• 影响因子: 7.6
• 作者: Mitra B;Thapa RJ;Guo H;Block TM
• 通讯作者: Block TM

Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication.

• DOI: 10.1038/srep39901
• 发表时间: 2017-01-03
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Pang J;Zhang G;Lin Y;Xie Z;Liu H;Tang L;Lu M;Yan R;Guo H;Sun J;Hou J;Zhang X
• 通讯作者: Zhang X

Hepatitis B virus X protein crosses out Smc5/6 complex to maintain covalently closed circular DNA transcription.

• DOI: 10.1002/hep.28834
• 发表时间: 2016-12
• 期刊: HEPATOLOGY
• 影响因子: 13.5
• 作者: Mitra, Bidisha;Guo, Haitao
• 通讯作者: Guo, Haitao

Epigenetic regulation of hepatitis B virus covalently closed circular DNA: Implications for epigenetic therapy against chronic hepatitis B.

• DOI: 10.1002/hep.29479
• 发表时间: 2017-12
• 期刊: Hepatology (Baltimore, Md.)
• 作者: Hong X;Kim ES;Guo H
• 通讯作者: Guo H

Biogenesis and molecular characteristics of serum hepatitis B virus RNA.

血清乙型肝炎病毒 RNA 的生物发生和分子特征。

• DOI: 10.1371/journal.ppat.1008945
• 发表时间: 2020-10
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Shen S;Xie Z;Cai D;Yu X;Zhang H;Kim ES;Zhou B;Hou J;Zhang X;Huang Q;Sun J;Guo H
• 通讯作者: Guo H