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

项目摘要和相关性。 乙肝病毒感染是一个世界性的健康问题。据估计，有2亿到5亿 对于世界上慢性乙肝病毒携带者，到目前为止，还没有可靠的治疗方法。乙肝病毒导致急性和 该病是一种慢性肝病，估计每年造成100万人死亡。目前可用 治疗方法可以减少病毒载量，但不能解决慢性乙肝病毒感染。因此，慢性阻塞性肺疾病的有效治疗 乙肝病毒感染已迫在眉睫。解决慢性乙肝病毒感染的主要障碍是 病毒模板--核共价闭合环状DNA的根除或灭活 抄写。为此，我们建立了hnf1α缺失的乙肝转基因小鼠和肝脏特异的tet缺陷小鼠。 乙肝转基因小鼠。HNF1α缺失的乙肝转基因小鼠合成核HBVccDNA，其中一部分是 在成年小鼠体内广泛甲基化。肝脏特异的Tet缺陷的乙肝转基因小鼠基本上是活的 肝脏生理正常，但缺乏可检测到的乙肝病毒转录和复制(即它们有效 “治愈”)表明Tet对病毒的生物合成也是必不可少的。Tet缺陷型乙肝病毒转基因的观察 小鼠不支持乙肝病毒的生物合成与Tet对发育至关重要的提示是一致的 乙肝病毒基因组DNA去甲基化，通过调节病毒的表观遗传方式调控乙肝病毒转录 体内染色质结构。确定与乙肝病毒相关的Tet表达的精确时间要求 转录和复制将指示肝脏发育阶段，此时病毒生物合成容易感染 Tet缺乏所致的抑制。这将通过使用我们最近开发的Tet表达调制来实现 他莫昔芬诱导的Tet缺陷乙肝转基因小鼠模型系统。使用该系统，开发的 将建立通过Tet表达控制乙肝病毒转录、病毒生物合成和HBVDNA甲基化的方法 并与与乙肝病毒基因组相关的表观遗传组蛋白标记和染色质结构相关。 类似的研究将使用HNF1Tet缺失型α转基因小鼠模型进行。 病毒感染对乙肝病毒转录、病毒生物合成、HBVDNA甲基化、 表观遗传组蛋白标记和染色质结构受Tet表达影响可与乙肝病毒基因组 乙肝病毒转基因DNA与核内HBVccDNA的比较最后，乙肝病毒的发展 支持病毒生物合成的转基因小鼠，完全来自染色体外基因组DNA， 生理相关的小鼠慢性病毒感染模型的建立，将会对Tet虚证的影响 在乙肝病毒的生物合成上，可以检测到单独来自于乙肝病毒的cccDNA。定义分子信号 Tet缺乏导致乙肝病毒生物合成丧失的责任可能导致细胞鉴定 治疗靶点，包括但不限于Tet蛋白，可用于开发新的 小分子治疗性抑制剂用于解决而不是简单地治疗慢性乙肝病毒感染。

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.