Developmental regulation of HBV biosynthesis by Ten-eleven translocation (Tet) methylcytosine dioxygenases

项目摘要和相关性。 B型肝炎病毒（HBV）感染是一个世界性的健康问题。据估计有2亿到5亿 HBV慢性携带者在世界上，迄今为止，没有可靠的治疗。HBV引起急性和 慢性肝病，每年估计造成一百万人死亡。当前可用 治疗降低病毒载量，但不能解决慢性HBV感染。因此，有效的治疗慢性 HBV感染是迫切需要的。解决慢性HBV感染的主要障碍是 根除或灭活核HBV共价闭合环状（ccc）DNA，其是病毒的模板， 转录。为此，我们开发了HNF 1 α-null HBV转基因小鼠和肝脏特异性Tet-deficient HBV转基因小鼠。HNF 1 α-null HBV转基因小鼠合成核HBV cccDNA，其中一部分是 在成年小鼠中广泛甲基化。肝脏特异性Tet-deficient HBV转基因小鼠是可行的，基本上 显示正常的肝脏生理学，但缺乏可检测的HBV转录和复制（即它们有效地 “治愈”），表明泰特也是病毒生物合成所必需的。Tet-deficient HBV转基因 小鼠不能支持HBV的生物合成与泰特对发育过程至关重要的建议一致。 HBV基因组DNA的去甲基化，其通过调节病毒表达而在表观遗传学上控制HBV转录 体内染色质结构。定义HBV相关泰特表达的精确时间要求 转录和复制将指示当病毒生物合成易受感染时的肝脏发育阶段。 泰特缺乏抑制。这将通过调节泰特表达来实现，使用我们最近开发的 他莫昔芬诱导Tet-deficient HBV转基因小鼠模型系统。利用这一系统， 通过泰特表达控制HBV转录、病毒生物合成和HBV DNA甲基化 并与HBV基因组相关的表观遗传组蛋白标记和染色质结构相关。 将使用HNF 1 α-null Tet-deficient HBV转基因小鼠模型进行类似的研究。 因此，HBV转录、病毒生物合成、HBV DNA甲基化， 通过与HBV基因组相关的泰特表达的表观遗传组蛋白标记和染色质结构， 比较HBV转基因DNA和核HBV cccDNA。最后，HBV的发展 转基因小鼠支持病毒生物合成完全从染色体外基因组DNA，一个更 生理相关的慢性病毒感染的小鼠模型，将制定这样的效果Tet-deficiency 可以确定对仅来源于HBV cccDNA的HBV生物合成的影响。定义分子信号 由于泰特缺乏导致HBV生物合成丧失的原因可能导致细胞内 治疗靶点，包括但不限于泰特蛋白，其适合于开发新的治疗靶点。 小分子量的治疗性抑制剂，以解决而不是简单地治疗慢性HBV感染。

Project Summary and Relevance. Hepatitis B virus (HBV) infection is a worldwide health problem. It is estimated that there are 200 to 500 million HBV chronic carriers in the world for whom, to date, there is no reliable treatment. HBV causes both acute and chronic liver disease and is responsible for an estimated one million deaths annually. Currently available therapies reduce viral loads but fail to resolve chronic HBV infections. Therefore, effective treatments for chronic HBV infection are urgently required. The major obstacle to the resolution of chronic HBV infections is the eradication or inactivation of nuclear HBV covalently closed circular (ccc) DNA which is the template for viral transcription. To this end, we have developed HNF1α-null HBV transgenic mice and liver-specific Tet-deficient HBV transgenic mice. HNF1α-null HBV transgenic mice synthesize nuclear HBV cccDNA, a fraction of which is extensively methylated in adult mice. Liver-specific Tet-deficient HBV transgenic mice are viable, essentially display normal liver physiology, but lack detectable HBV transcription and replication (i.e. they are effectively “cured”) suggesting Tet is also essential for viral biosynthesis. The observation that Tet-deficient HBV transgenic mice fail to support HBV biosynthesis is consistent with the suggestion that Tet is essential for the developmental demethylation of HBV genomic DNA which epigenetically governs HBV transcription by modulating viral chromatin structure in vivo. Defining the precise temporal requirements for Tet expression associated with HBV transcription and replication will indicate the liver developmental stages when viral biosynthesis is susceptible to inhibition by Tet deficiency. This will be achieved by modulating Tet expression using our recently developed tamoxifen-inducible Tet-deficient HBV transgenic mouse model system. Using this system, the developmental control of HBV transcription, viral biosynthesis and HBV DNA methylation by Tet expression will be established and correlated with the epigenetic histone marks and chromatin structure associated with the HBV genome. Similar studies will be performed using the HNF1α-null Tet-deficient HBV transgenic mouse model of chronic viral infection so the developmental control of HBV transcription, viral biosynthesis, HBV DNA methylation, epigenetic histone marks and chromatin structure by Tet expression associated with the HBV genome can be compared between the HBV transgene DNA and the nuclear HBV cccDNA. Finally, the development of HBV transgenic mice supporting viral biosynthesis exclusively from extrachromosomal genomic DNA, a more physiologically relevant mouse model of chronic viral infection, will be developed so the effect of Tet-deficiency on HBV biosynthesis derived solely from HBV cccDNA can be determined. Defining the molecular signals responsible for the loss of HBV biosynthesis due to Tet deficiency may lead to the identification of cellular therapeutic targets, including but not restricted to Tet proteins, that are amenable to the development of novel small molecular weight therapeutic inhibitors to resolve rather than simply treat chronic HBV infection.