MECHANISM OF ACTION OF HIV REGULATORY PROTEINS

描述：(改编自申请者摘要)本项目的目标 是为了研究HIV TAT(反式激活因子)的细胞控制机制 基因的功能。重点放在一组独特的人类宿主细胞核上 特异性识别TAT靶标的茎区域的因子 TAR RNA序列。对TAR RNA特定突变体的研究表明 宿主因子体外结合与体内TAT功能相关 这种宿主细胞调节的反式激活的效率取决于 蛋白激酶C(PKC)。这个项目的具体目的是尝试 为了了解与有丝分裂诱导有关的分子机制 病毒基因反式激活的宿主细胞。拟议中的实验， 利用亲和层析和免疫化学鉴定， 旨在了解激酶和磷酸酶的特异性， 调节宿主细胞TAT共因子的功能相互作用。其他 程序将尝试分子克隆和表达人的cdna TAT辅助因子的编码序列及其作用机制研究 对病毒激活的调节。无细胞重组转录 将利用系统来破译TAT依赖的性质 HIV LTR(Long)控制下的基因转录激活 末端重复)，以及宿主细胞TAT辅助因子在 这一过程的监管。TAR靶标的定点突变体 序列和宿主细胞TAT辅助因子将以类似的方式分析 重组系统以解决其分子的性质 宿主细胞调控的相互作用及其功能意义 病毒基因反式激活。

DESCRIPTION: (Adapted from applicant's abstract) The goal of this project is to study the mechanism of cellular control of HIV tat (transactivator) gene function. The focus is on a unique set of human host cell nuclear factors that specifically recognize the stem region of the Tat target sequence TAR RNA. Studies with specific mutants of TAR RNA showed that host factor binding in vitro correlates with Tat function in vivo and that the efficiency of this host cell regulated trans-activation is dependent upon protein kinase C (PKC). The specific aim of this project is to try to understand the molecular mechanisms that link mitogenic induction of the host cell with viral gene trans-activation. The proposed experiments, using affinity chromatography and immunochemical characterization, are designed to understand the specificity of kinases and phosphatases that regulate functional interaction of the host cell Tat co-factor. Other procedures will attempt molecular cloning and expression of human cDNA sequences encoding the Tat co-factor and study of the mechanism of its regulation of viral activation. Cell-free reconstituted transcription systems will be utilized to decipher the nature of Tat-dependent transcriptional activation of genes under the control of the HIV LTR (long terminal repeat), and the specific role host cell Tat co-factor plays in the regulation of the process. Site-directed mutants of the TAR target sequences and the host cell Tat co-factor will be analyzed in similar reconstituted systems to resolve the nature of their molecular interactions and the functional significance of the host cell modulated viral gene trans-activation.