MECHANISM OF ACTION OF HIV REGULATORY PROTEIN

描述：（改编自申请人的摘要）本项目的目标 是研究HIV达特（反式激活因子）的细胞控制机制 基因功能 重点是一套独特的人类宿主细胞核 特异性识别达特靶的茎区的因子 序列TAR RNA。 对TAR RNA特定突变体的研究表明， 体外宿主因子结合与体内达特功能相关， 这种宿主细胞调节的反式激活的效率依赖于 蛋白激酶C（PKC）。 该项目的具体目标是尝试 为了了解有丝分裂诱导的分子机制， 具有病毒基因反式激活的宿主细胞。 拟议的实验， 使用亲和层析和免疫化学表征， 旨在了解激酶和磷酸酶的特异性， 调节宿主细胞达特辅因子的功能相互作用。 其他 程序将尝试分子克隆和表达人类cDNA 达特辅因子的编码序列及其作用机制的研究 调节病毒活化。 无细胞重组转录 系统将被用来破译依赖Tat的性质， 在HIV LTR控制下的基因的转录激活（长 末端重复序列），以及宿主细胞达特辅因子在 过程的规范。 TAR靶标的定点突变体 序列和宿主细胞达特辅因子将以类似的方式进行分析。 重组系统，以解决其分子的性质， 调节的宿主细胞的相互作用和功能意义 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.