HIV 1 LTR REGULATORY ELEMENTS

HIV-1基因的表达受多种细胞因子的调控 结合顺式作用调控元件的转录因子 HIV-1 LTR.这些细胞因子与病毒之间的相互作用 反式激活蛋白TAT对基因调控至关重要。除了……之外 TAT等多种调节因子对HIV-1基因表达的调控 通过信号转导途径促进HIV-1基因的表达 改变细胞转录因子的活性。它是 上游转录因子的相互作用，一般转录因子 机制，以及导致HIV-1基因表达激活的TAT。 这项拨款提案的重点是更好地理解复杂的 导致HIV-1复制增加的转录机制。 特别是，我们希望进一步分析顺式代理监管 影响病毒复制的因素，使用生化 用于鉴定细胞转录因子的分级技术 HIV-1基因调控，并利用体外转录检测 剖析TAT所必需的转录途径的组成部分 激活。我们产生含有定点定向的病毒的能力 在每个关键的顺式作用HIV-1 LTR元件中的突变体， TAT诱导的固定化HIV-1模板检测方法的建立 HIV-1 LTR体外转录水平的提高及纯化 新的和先前描述的普通和上游细胞 转录因子为我们提供了技术和试剂 对于更好地理解TAT激活是必要的。这样做的具体目的是 建议：(L)使用与链霉亲和素结合的固定化艾滋病毒-1模板 用于剖析转录途径中被改变的步骤的珠子 TAT和细胞调节蛋白；(2)更好地表征 HIV-1中特定序列和细胞因子的作用机制 TATA元件影响基因表达的激活；(3)确定如何 RNA聚合酶II的转录延伸特性为 被细胞因子和TAT修饰；(4)了解差异 HIV-1 LTR调节区的突变改变了病毒的复制和 胸腺重组SCID-Hu小鼠模型的后续发病机制 以及(5)确定细胞的体内结合特性如何 HIV-1 LTR的因子在含有突变的病毒中发生变化 Tat基因或在不同的HIV-1 LTR调控区。这些 研究将有助于解决调节细胞因子的作用 HIV-1基因的表达。

HIV-1 gene expression is regulated by a diverse set of cellular transcription factors which bind to cis-acting regulatory elements in the HIV-1 LTR. The interplay between these cellular factors and the viral transactivator protein Tat is critical for gene regulation. In addition to modulation of HIV-1 gene expression by Tat, a variety of other regulators of the signal transduction pathway can increase HIV-1 gene expression by altering the activity of cellular transcription factors. It is the interaction of upstream transcription factors, the general transcriptional machinery, and Tat which results in activation of HIV-1 gene expression. The focus of this grant proposal is to better understand the complex transcriptional machinery that results in increases in HIV-1 replication. In particular, we wish to further analyze the cis-acting regulatory elements that influence viral replication, to use biochemical fractionation to identify cellular transcription factors important for HIV-1 gene regulation, and to utilize in vitro transcription assays to dissect components of the transcriptional pathway necessary for Tat activation. Our ability to produce viruses containing site-directed mutants in each of the critical cis-acting HIV-1 LTR elements, the development of immobilized HIV-1 template assays which give Tat induced increases in in vitro transcription from HIV-1 LTR, and the purification of both novel and previously characterized general and upstream cellular transcription factors provides us with the techniques and reagents necessary to better understand Tat activation. The specific aims of this proposal are: (l) to use immobilized HIV-1 templates bound to streptavidin beads to dissect steps in the transcriptional pathway that are altered by Tat and cellular regulatory proteins; (2) to better characterize the mechanism by which specific sequences and cellular factors in the HIV-1 TATA element influence activation of gene expression; (3) to determine how the transcriptional elongation properties of RNA polymerase II are modified by cellular factors and Tat; (4) to understand how different mutations in HIV-1 LTR regulatory regions alter viral replication and subsequent pathogenesis in the thymus reconstituted SCID-hu mouse model and (5) to determine how the in vivo binding properties of cellular factors to the HIV-1 LTR are altered in viruses containing mutations in either the tat gene or in different HIV-1 LTR regulatory regions. These studies will help to address the role of cellular factors which modulate HIV-1 gene expression.