Developmental regulation of HBV biosynthesis by FoxA and DNA methylation

乙肝病毒感染是一个世界性的健康问题。据估计，世界上有2亿至5亿慢性乙肝病毒携带者，到目前为止，还没有可靠的治疗方法。乙肝病毒会导致急性和慢性肝病，每年估计造成100万人死亡。目前可用的治疗方法可以减少病毒载量，但无法解决慢性乙肝病毒感染。因此，迫切需要对慢性乙肝进行有效的治疗。慢性乙肝病毒感染解决的主要障碍是作为病毒转录模板的核型乙肝病毒共价闭合环(CCC)DNA的根除或失活。为此，我们建立了HNF1α缺失的乙肝转基因小鼠和肝脏特异的FoxA/HNF3缺陷的乙肝转基因小鼠。HNF1HBVccDNA缺失的转基因小鼠合成α核心DNA。肝脏特异的FoxA/HNF3缺陷的HBV转基因小鼠是存活的，但缺乏可检测到的HBV转录和复制(即它们被有效地“治愈”)，这表明FoxA/HNF3不仅在控制肝脏表型的肝细胞特异转录因子网络中发挥着重要的发育作用，而且对病毒的生物合成也是必不可少的。FoxA/HNF3基因缺陷的乙肝病毒转基因小鼠的基因组DNA高甲基化的观察表明，FoxA/HNF3在体内通过调节病毒染色质结构来调控乙肝病毒的转录，从而决定病毒的生物合成水平。确定与乙肝病毒转录和复制相关的FoxA/HNF3表达的准确时间要求将指示肝脏发育阶段，此时病毒生物合成容易受到FoxA/HNF3缺陷的抑制。这将通过使用Tet/Off系统在FoxA/HNF3缺陷的乙肝转基因小鼠模型中调节FoxA/HNF3的表达来实现。利用该系统，将建立通过FoxA/HNF3表达对乙肝病毒转录、病毒生物合成和HBVDNA甲基化的发育控制，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联。最后，将使用HNF1FoxA/HNF3缺失的慢性病毒感染的α转基因小鼠模型进行类似的研究，以便比较与HBVDNA基因组相关的FoxA/HNF3表达对HBVDNA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的发育控制。确定这些变化的性质和由于FoxA/HNF3缺乏导致乙肝病毒生物合成丧失的分子信号可能导致识别细胞治疗靶点，这些靶点有助于开发新的方法来解决而不是简单地治疗慢性乙肝感染。

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 FoxA/HNF3-deficient HBV transgenic mice. HNF1α-null HBV transgenic mice synthesize nuclear HBV CCC DNA. Liver-specific FoxA/HNF3-deficient HBV transgenic mice are viable but lack detectable HBV transcription and replication (i.e. they are effectively “cured”) suggesting FoxA/HNF3 not only plays an important developmental role in the hepatocyte-specific transcription factor network governing the hepatic phenotype but is also essential for viral biosynthesis. The observation that FoxA/HNF3-deficient HBV transgenic mice display hyper-methylation of HBV genomic DNA suggests that FoxA/HNF3 is epigenetically governing HBV transcription by modulating viral chromatin structure in vivo and hence determines the level of viral biosynthesis. Defining the precise temporal requirements for FoxA/HNF3 expression associated with HBV transcription and replication will indicate the liver developmental stages when viral biosynthesis is susceptible to inhibition by FoxA/HNF3 deficiency. This will be achieved by modulating FoxA/HNF3 expression in the FoxA/HNF3-deficient HBV transgenic mouse model using the TET/OFF system. Using this system, the developmental control of HBV transcription, viral biosynthesis and HBV DNA methylation by FoxA/HNF3 expression will be established and correlated with the epigenetic histone marks and chromatin structure associated with the HBV genome. Finally, similar studies will be performed using the HNF1α-null FoxA/HNF3-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 FoxA/HNF3 expression associated with the HBV genome can be compared between the HBV transgene DNA and the nuclear HBV CCC DNA. Defining the nature of the changes and the molecular signals responsible for the loss of HBV biosynthesis due to FoxA/HNF3 deficiency may lead to the identification of cellular therapeutic targets that are amenable to the development of novel modalities to resolve rather than simply treat chronic HBV infection.