TRANSCRIPTIONAL REGULATION OF HTLV-GENE EXPRESSION

本建议中所述研究的目的是为了更充分地了解 了解人类T细胞调节的机制 白血病病毒（HTLV-I）基因表达。 除了结构 HTLV-I编码多种调节病毒基因的蛋白质， 在转录水平上表达。 Tax 1是其中一种蛋白质， 病毒基因表达的40 kD阳性反式激活因子。 出租车是独一无二的 与其他转录调节因子不同， 不能直接与DNA结合。 但是，Tax 1可以关联 通过细胞转录因子与DNA间接连接。 的这种机制 与DNA间接结合使人想起其他几种转录 包括腺病毒ElA和疱疹病毒VP 16的因子。 这些属性， 以及缺乏典型的酸性激活结构域，表明 Taxi可以与转录共激活因子/衔接子相互作用。 在 提出的研究，转录激活的分子机制， 将对Tax 1进行研究，特别关注可能的相互作用 与细胞转录因子结合。(i)Tax 1的激活结构域， 将通过构建含有已知DNA的嵌合蛋白质来定义 与Tax 1蛋白或各种Tax 1融合的结合结构域（GA 14，aa 1-147）， 变种人 这些融合蛋白将被测试它们的能力， 反式激活LTR-CAT报告基因构建体，其中特异性Taxi应答 元件已被GAL 4结合位点取代。(ii)细胞蛋白 将分离并研究能够与Tax 1相互作用的细胞，以确定 如果它们在Tax 1反式激活中发挥特定作用。(iii)体外 将开发转录系统，以研究 Tax 1反式激活。(iv)Tax 1的晶体结构为 为了将功能结构域与特定结构域相关联， 蛋白质的特征。 由于Tax 1不具有任何强同源性， 与已知的转录激活结构域，它是一个理想的候选人， 揭示了调节这些功能的新结构。 总体上 这些研究所产生的见解将提供一个更完整的 了解真核基因调控的机制。 具体来说，这些结果将加强我们对HTLV-I潜伏期的了解 和转化，可能导致治疗进展， HTLV-I相关疾病，包括成人T细胞白血病和热带 痉挛性麻痹-HTLV-1相关的脊髓病。

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.