REGULATION OF HEPATITIS B VIRUS TRANSCRIPTION

由B型肝炎病毒（HBV）引起的公共卫生问题是 在世界范围内的重要性，因为在远东亚洲的慢性携带者状态 热带非洲占人口的10%或更多， 活动性肝炎和肝硬化是死亡的主要原因。 HBV 一般来说，接种疫苗可预防感染。 然而，可靠 治疗约2亿HBV慢性携带者 目前全球范围内都没有。 消除复制 从慢性携带者中分离病毒可以通过抑制病毒 转录，这是病毒复制周期中的重要步骤。 因此，详细了解调节 HBV转录单位的表达可能提示合理的 破坏病毒生命周期中这一步骤的方法。 3.2kb HBV基因组中与HBV DNA序列相关的核苷酸序列元件 调节这四种病毒转录单位的表达将是 在各种肝脏和非肝脏细胞系中使用瞬时 转染测定和体外转录系统。 一系列 将构建主要表面抗原的启动子缺失， 大表面抗原、核衣壳抗原和X基因，并检查 通过测量RNA水平和/或报告基因的转录活性 由这些调节序列元件指导的活动。 详细 这些基因的转录调控元件的检查 将通过表征集群和单个 HBV启动子活性的点突变。 的因素 存在于各种细胞提取物中，其结合HBV调节蛋白， 将使用凝胶阻滞和Dnasel足迹法检查序列 测定。 转录分析和DNA结合的结果 将比较测定以确定这两种测定之间的任何相关性。 活动 这一分析可能表明， 与肝细胞特异性转录相互作用的序列元件 因此，可能有助于HBV肝嗜性。 序列 HBV启动子转录调节所必需的元件 糖皮质激素和转化生长因子β的活性也将 接受检查。 定义HBV启动子调控序列元件， 序列特异性DNA结合因子的检测代表了 进一步鉴定和克隆基因编码 这些转录因子。 克隆这些因子的基因 将允许分析这些因素的机制 与它们的识别序列相互作用， HBV启动子的转录活性。

The public health problem caused by hepatitis B virus (HBV) is of worldwide importance since the chronic carrier state in Far East Asia and tropical Africa represents 10% or more of the population and chronic active hepatitis and liver cirrhosis are major causes of mortality. HBV infection can generally be prevented by vaccination. However, reliable treatments for the approximately 200 million HBV chronic carriers worldwide are currently unavailable. The elimination of replicating virus from chronic carriers might be achieved by inhibiting viral transcription, an essential step in the replication cycle of the virus. Therefore, a detailed understanding of the mechanisms regulating the expression of the HBV transcription units may suggest rational approaches to disrupting this step in the viral life cycle. The nucleotide sequence elements in the 3.2kb HBV genome involved in regulating the expression of the four viral transcription units will be examined in a variety of liver and non-liver cell lines using transient transfection assays and in vitro transcription systems. A series of promoter deletions will be constructed for the major surface antigen, large surface antigen, nucleocapsid antigen and X genes and examined for transcriptional activity by measuring RNA levels and/or reporter gene activities directed by these regulatory sequence elements. A detailed examination of the transcriptional regulatory elements of these genes will be performed by characterizing the effects of clustered and single point mutations on the activities of the HBV promoters. The factors present in various cell extracts that bind to the HBV regulatory sequences will be examined using gel retardation and Dnasel footprinting assays. The results of the transcriptional analysis and the DNA binding assays will be compared to determine any correlations between these two activities. This analysis may indicate the presence of regulatory sequence elements which interact with hepatocyte specific transcription factors and therefore may contribute to HBV liver tropism. The sequence elements necessary for transcriptional modulation of the HBV promoter activities by glucocorticoids and transforming growth factor p will also be examined. Defining the HBV promoter regulatory sequence elements and detection of sequence specific DNA binding factors represents an initial step towards the characterization and cloning of the genes encoding these transcription factors. The cloning of the genes for these factors will permit the analysis of the mechanism by which these factors interact with their recognition sequences and influence the transcriptional activity of the HBV promoters.