MECHANISM OF ACTION OF THE HIV REGULATORY PROTEINS

人类免疫缺陷病毒（HIV，原HTLVIII/LAV/ARV） 是获得性免疫缺陷的病原体 综合征（艾滋病）及相关疾病。 在人类中， 感染明显表现出潜伏期延长， 散布着病毒复制爆发的复制水平 以及伴随的T细胞死亡。 虽然病毒的机制 潜伏期是未知的，最近的报告表明， 病毒复制所需的基因产物， 对于从潜伏期到细胞病变期的转变至关重要 病毒感染。 我们的研究将集中在生物化学和 这两种反式激活因子的分子遗传学分析 蛋白质，达特III（HTLV/III反式激活基因）和art（抗- 阻遏反式激活因子），以及它们调节 HIV基因表达。 直接分析他们的目标部位 结合和相关的研究，我们已经克隆了达特III， 将本发明的蛋白质编码序列克隆到细胞中的方法 使用杆状病毒的有效真核表达载体系统 vector. 达特III与TAR结合的作用将在 在无细胞翻译系统中体外合成mRNA。 类似地，达特III在mRNA稳定化中的作用将是 使用无细胞转录系统研究， HIV模板DNA和克隆的达特III蛋白。 类似 重构的细胞自由翻译和剪接系统， 并辅以艺术基因产品，分析其作用机理 HIV gag和env基因的转录后调节 由art蛋白质转录。 后续实验将聚焦 定点诱变以确定所述酶的活性位点。 克隆的达特III和art蛋白，从而更好地定义它们的 作用机制。 因此，这些研究将描述 两个重要的生物化学和生物物理参数 病毒调节蛋白，从而帮助确定其机制， 在诱导HIV感染的细胞病变阶段中的作用， 人类 所获得的知识对我们的设计很重要， 遏制艾滋病毒感染和艾滋病的发展。

Human immunodeficiency virus (HIV formerly HTLVIII/LAV/ARV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and associated diseases. In humans, HIV infection apparently displays a prolonged latent period with low levels of replication interspersed with bursts of viral replication and concomittant T cell death. Although the mechanism of viral latency is unknown, recent reports have implicated specific HIV gene products that are required for viral replication and thus may be essential for the shift from the latent to the cytopathic phase of viral infection. Our studies will focus on the biochemical and molecular genetic analysis of the two such trans-activator proteins, tat III (HTLV/III trans-activating gene) and art (anti- repression transactivator), and the modes of their regulation of HIV gene expression. For direct analysis of their target site binding and related studies, we have cloned the tat III and are in the process of cloning the art protein coding sequences in an efficient eukaryotic expression vector system using a baculovirus vector. The role of tat III binding to TAR will be studied using in vitro synthesized mRNAs in a cell free translation system. Similarly, the role of tat III in stabilization of mRNA will be studied using cell-free transcription system supplemented with HIV template DNA and cloned tat III protein. Similar reconstituted cell free translation and splicing systems, supplemented with art gene product, will analyze the mechanism of post-transcriptional regulation of the HIV gag and env gene transcripts by the art protein. Subsequent experiments will focus on site directed mutagenesis to determine the active sites of the cloned tat III and art proteins, and thus better define their mechanism of action. These studies therefore will delineate the biochemical and biophysical parameters of the two important viral regulatory proteins and thus help define their mechanisms of action in inducing the cytopathic phase of HIV infection in humans. The knowledge gained will be important in our designs to contain HIV infection and the progression of AIDS.