Developmental regulation of HBV biosynthesis by FoxA and DNA methylation

B型肝炎病毒（HBV）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HBV慢性携带者，迄今为止，没有可靠的治疗方法。HBV引起急性和慢性肝病，每年估计造成100万人死亡。目前可用的疗法降低病毒载量，但未能解决慢性HBV感染。因此，迫切需要有效治疗慢性HBV感染。解决慢性HBV感染的主要障碍是消除或灭活作为病毒转录模板的核HBV共价闭合环状（CCC）DNA。为此，我们开发了HNF 1 α缺失型HBV转基因小鼠和肝脏特异性FoxA/HNF 3缺陷型HBV转基因小鼠。HNF 1 α基因敲除的HBV转基因小鼠合成核HBV CCC DNA。肝脏特异性FoxA/HNF 3缺陷型HBV转基因小鼠是可行的，但缺乏可检测的HBV转录和复制（即它们被有效地“治愈”），这表明FoxA/HNF 3不仅在控制肝脏表型的肝细胞特异性转录因子网络中发挥重要的发育作用，而且对病毒生物合成也是必需的。观察到FoxA/HNF 3缺陷型HBV转基因小鼠显示HBV基因组DNA的超甲基化，这表明FoxA/HNF 3通过调节体内病毒染色质结构而在表观遗传学上控制HBV转录，因此决定了病毒生物合成的水平。定义与HBV转录和复制相关的FoxA/HNF 3表达的精确时间要求将指示当病毒生物合成易受FoxA/HNF 3缺陷抑制时的肝脏发育阶段。这将通过使用泰特/OFF系统调节FoxA/HNF 3缺陷型HBV转基因小鼠模型中的FoxA/HNF 3表达来实现。使用该系统，将建立通过FoxA/HNF 3表达对HBV转录、病毒生物合成和HBV DNA甲基化的发育控制，并将其与HBV基因组相关的表观遗传组蛋白标记和染色质结构相关联。最后，将使用慢性病毒感染的HNF 1 α-null FoxA/HNF 3缺陷型HBV转基因小鼠模型进行类似的研究，因此可以在HBV转基因DNA和核HBV CCC DNA之间比较与HBV基因组相关的FoxA/HNF 3表达对HBV转录、病毒生物合成、HBV DNA甲基化、表观遗传组蛋白标记和染色质结构的发育控制。定义的性质的变化和负责的HBV生物合成的损失，由于FoxA/HNF 3缺陷的分子信号可能会导致识别的细胞治疗靶点，适合于开发新的方式来解决，而不是简单地治疗慢性HBV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.