REGULATION OF HIV GENE EXPRESSION BY TAT MUTANT PEPTIDE

HIV-1编码一种反式激活蛋白Tat，可激活病毒 基因表达，是艾滋病毒复制所必需的，因此是一种 艾滋病治疗发展的潜在靶点。我们的主要目标 是了解TAT结构与功能的关系，研究 TAT传输和翻译后修改，以确定 显性负性突变TAT多肽作为潜在的TAT拮抗剂， 并开发提供和测试TAT有效性的方法 作为艾滋病毒表达抑制物的多肽拮抗剂。我们有 建立哺乳动物细胞微量注射测定TAT的方法 并在化学上合成了生物 活性全长86个氨基酸的TAT蛋白和几个缺失 编码结构域的多肽具有高反式激活活性。 我们建议准确地划定这些领域，以便于 结构-功能研究及开发黄曲霉多肽拮抗剂 TAT功能。单一氨基酸的替代将在 最小TAT结构域肽。这将允许识别 的必需氨基酸存在，并可导致TAT的拮抗剂 功能。目前已鉴定出几种Tat突变多肽 以这种方式抑制野生型TAT的反式激活。 我们将通过增加氨基进一步开发TAT突变拮抗剂 酸替代并测试其抑制HIV的能力 T淋巴细胞中的复制，作为其潜力的指示 用于艾滋病的治疗。结构-功能要求 TAT多肽在T淋巴细胞中的摄取和核转运 将会被研究。合成编码TAT拮抗剂多肽的基因 将被克隆到腺病毒载体中用于感染HIV 感染T4淋巴细胞作为TAT多肽疗效试验的研究 对抗者。这些载体也可能用于艾滋病的临床治疗 心理治疗。作为一个长远的目标，我们将开发表达载体 基于TAT多肽反式激活调控HIV-LTR的系统 基因。

HIV-1 encodes a transactivating protein, tat, that activates viral gene expression, is essential for HIV replication, and thus is a potential target for development of AIDS therapy. Our major goals are to understand tat structure-function relationships, to study tat transport and posttranslation modifications, to identify dominant negative mutant tat peptides as potential tat antagonists, and to develop methods to deliver and test the efficacy of tat peptide antagonists as inhibitors of HIV expression. We have developed a mammalian cell microinjection assay for tat transactivation and have chemically synthesized the biologically active full length 86 amino acid tat protein and several deletion peptides encoding domains possessing high transactivating activity. We propose to precisely delineate these domains to facilitate structure-function studies and to develop peptide antagonists of tat function. Single amino acid substitutions will be made in minimal tat domain peptides. This will allow the identification of essential amino acid resides and can lead to antagonists of tat function. Several tat mutant peptides have already been identified in this manner which inhibit transactivation by wild type tat. We will further develop tat mutant antagonists by additional amino acid substitutions and test their ability to inhibit HIV replication in T lymphocytes, as an indication of their potential use for therapy of AIDS. Structure-function requirements for uptake and nuclear transport of tat peptides into T lymphocytes will be studied. Synthetic genes encoding tat antagonist peptides will be cloned into adenovirus vectors for infection of HIV infected T4 lymphocytes as a test of the efficacy of tat peptide antagonists. These vectors may also have clinical use for AIDS therapy. As a long range goal, we will develop expression vector systems based on tat peptide transactivation of HIV-LTR regulated genes.