TRANSCRIPTIONAL REGULATION OF HTLV-GENE EXPRESSION

这项建议中描述的研究的目的是为了获得更全面的 对人类T细胞调控机制的认识 白血病病毒(HTLV-I)基因表达。除了结构 蛋白质，HTLV-I编码几种调节病毒基因的蛋白质 在转录水平上表达。其中一种蛋白质Tax1是一种 40kD阳性病毒基因表达反式激活因子。出租车独一无二 来自许多其他转录调控者，因为它似乎是 不能直接与DNA结合的。但是，Tax1可以关联 通过细胞转录因子间接与DNA结合。这一机制 与DNA的间接关联让人联想到其他几种转录 因素包括腺病毒ELA和疱疹病毒VP16。这些属性， 以及没有经典的酸性激活结构域，这表明 出租车可能与转录辅活化子/适配器相互作用(S)。在 建议的研究，转录激活的分子机制 将对可能的相互作用进行特别感兴趣的研究 与细胞转录因子结合。(I)Tax1的激活结构域， 将通过构建包含已知DNA的嵌合蛋白来定义 结合结构域(GAl4，AA 1-147)与Tax1、蛋白质或各种Tax1融合， 变种人。这些融合蛋白将被测试它们是否有能力 反式激活LTR-CAT报告结构，其中特定的Taxi响应 元素已被GAL4结合位点取代。(Ii)细胞蛋白质 能够与Tax1相互作用的基因将被分离并研究以确定 如果它们在Tax1反式激活中起到特定的作用。(Iii)体外培养 将建立转录系统以研究其机制。 Tax1反式激活。(4)紫杉醇1的晶体结构为 确定是为了将功能结构域与特定的结构相关联 蛋白质的特性。由于Tax1不具有任何强同源 与已知的转录激活结构域，它是一个理想的候选者 揭示了调节这些功能的新结构。总体而言， 这些研究产生的见解将提供更完整的 对真核基因调控机制的理解。 具体地说，这些结果将加强我们对HTLV-I延迟的了解 和转化，可能导致治疗方面的进步 与HTLV-I相关的疾病，包括成人T细胞白血病和热带 痉挛截瘫-巨细胞病毒-L相关性脊髓病。

The purpose of the studies described in this proposal is to gain a fuller understanding of the mechanisms involved in regulation of human T cell leukemia virus (HTLV-I) gene expression. In addition to structural proteins, HTLV-I encodes several proteins which regulate viral gene expression at the level of transcription. One of these proteins, Tax1 is a 40 kD positive transactivator of viral gene expression. Taxi is unique from many other transcriptional regulators in that it appears to be incapable of binding directly to DNA. However, Tax1 can associate indirectly with DNA via a cellular transcription factor. This mechanism of indirect association with DNA is reminiscent of several other transcription factors including adenovirus ElA and herpesvirus VP16. These properties, as well as the absence of a classic acidic activating domain, suggest that Taxi may interact with a transcriptional coactivator/adaptor(s). In the proposed studies, the molecular mechanisms of transcription activation by Tax1 will be investigated with particular interest in possible interactions with cellular transcription factors. (i) The activation domain of Tax1, will be defined by constructing chimeric proteins containing a known DNA binding domain (GAl4, aa 1-147) fused to the Tax1, protein or various Tax1, mutants. These fusion proteins will be tested for their ability to transactivate LTR-CAT reporter constructs in which specific Taxi responsive elements have been replaced with GAL4 binding sites. (ii) Cellular proteins capable of interacting with Tax1 will be isolated and studied to determine if they play a specific role in Tax1 transactivation. (iii) An in vitro transcription system will be developed with which to study the mechanism of Tax1 transactivation. (iv) The crystal structure of Tax1 will be determinedin order to associate functional domains with specific structural features of the protein. Since Tax1 does not possess any strong homologies with known transcription activating domains, it is anideal candidate for revealing new structures which mediate these functions. In general, the insights generated by these studies will provide a more complete understanding of the mechanisms of eukaryotic gene regulation. Specifically, the results will strengthen our knowledge of HTLV-I latency and transformation, possibly resulting in therapeutic advances in treatment of HTLV-I associated diseases including adult T-cell leukemia and tropical spastic paraparesis-HTLV-l associated myelopathy.