TRANSCRIPTIONAL REGULATION BY HEPTITIS B VIRUS X PROTEIN

乙型肝炎病毒 X 蛋白的转录调控

• 批准号: 3200885
• 负责人: Robert Schneider
• 金额: $ 18.36万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3200885
• 关键词: DNA binding protein; DNA directed RNA polymerase; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell growth regulation; chimeric proteins; complementary DNA; enzyme induction /repression; gene expression; genetic library; genetic mapping; genetic promoter element; genetic transcription; genetic translation; hepatitis B virus group; immunofluorescence technique; molecular cloning; molecular genetics; mutant; peptides; protein biosynthesis; protein purification; transcription factor; transfection; viral carcinogenesis; virus protein; western blottings

The long-term objectives of this proposal are to understand the fundamental mechanisms by which human hepatitis B virus (HBV) regulates gene expression and contributes to the production of hepatocellular carcinoma (HCC). The specific aims of this proposal can be summarized as four main goals: (1) To precisely map the X protein domains involved in activation and repression of transcription by RNA polymerases II and III. (2) To investigate the role of X protein in regulating transcription and identify potential regulatory target proteins. (3) To investigate whether X protein activates transcription elements by acting directly at the promoter, whether it act on the central regulators of key signal transduction pathways, or both. (4) To identify cellular polypeptides that likely interact with X protein, some of which could be central mediators of transcriptional regulation and cell growth. The manner by which HBV participates in generation of HCC, the factors which contribute to liver disease during chronic infection and the ability of the virus to maintain persistent infections for considerable periods of time are all areas which are poorly understood. The transcriptional regulatory properties of the HBV X protein has therefore been potentially implicated in many of these processes. Experiments have been proposed to investigate the molecular level at which X proteins function and their mechanism of action. Previous studies demonstrated that X protein activates transcription by RNA polymerases II and III. Recent studies indicate that the X gene actually encodes two related polypeptides, a full- length translation product and an amino-truncated species. Experiments are outlined to fully characterize the multiple transcriptional activities of both X proteins (activation and repression), and to finely map the protein regions involved in activation of RNA polymerases II and III. To dissect the molecular level and key cellular proteins acted on by X protein, inducible and regulatable X protein constructs are described which will enable determination of the transcription at the level of the promoter, or through participation in signal transduction pathways, or both. Given the ability to modulate the transcriptional activity of different classes and types of promoters, and the apparent inability to bind DNA, it seems likely that the X proteins function by interacting with important cellular regulatory polypeptides. Several approaches have been outlined to investigate the potential association of X proteins with cellular polypeptides, including identification by co-immunoprecipitation. Attempts will also be made to clone interacting cellular polypeptides by using purified labeled X protein to screen a cDNA expression library prepared from human liver.

该提议的长期目标是了解基本 人肝炎病毒（HBV）调节基因表达的机制 并有助于产生肝细胞癌（HCC）。 这 该提案的具体目的可以总结为四个主要目标：（1） 精确映射参与激活和 RNA聚合酶II和III对转录的抑制。 （2）至 研究X蛋白在调节转录中的作用并确定 潜在的调节靶蛋白。 （3）研究X蛋白是否 通过直接在启动子上作用来激活转录元件， 它是否作用于钥匙信号转导的中央调节器 路径，或两者兼而有之。 （4）鉴定可能的细胞多肽 与X蛋白相互作用，其中一些可以是 转录调控和细胞生长。 HBV参与HCC的生成方式，这些因素 在慢性感染期间会导致肝病和能力 在相当长的一段时间内维持持续感染的病毒 时间是所有知名度不佳的领域。 转录 因此，HBV X蛋白的调节特性已可能是潜在的 与许多此类过程有关。 已经提出了实验 研究X蛋白及其其功能的分子水平 作用机理。 先前的研究表明X蛋白 通过RNA聚合酶II和III激活转录。 最近的研究 表明X基因实际上编码了两个相关的多肽，一个完整的 长度翻译产物和氨基截断的物种。 实验是 概述以充分表征 X蛋白（激活和抑制）都可以精细绘制蛋白质 参与RNA聚合酶II和III的区域。 剖析 由X蛋白起作用的分子水平和关键细胞蛋白， 描述了可诱导和可调节的X蛋白构建体 启用在启动子水平上确定转录，或 通过参与信号转导途径，或两者兼而有之。 鉴于能够调节不同的转录活性 启动子的类和类型，以及明显无法结合DNA X蛋白似乎通过与重要的相互作用来起作用 细胞调节多肽。 已经概述了几种方法 研究X蛋白与细胞的潜在关联 多肽，包括通过共免疫沉淀识别。 尝试 还将通过使用克隆相互作用的细胞多肽 纯化标记的X蛋白来筛选制备的cDNA表达式文库 来自人类肝脏。