HIV 1 LTR REGULATORY ELEMENTS

HIV-1基因表达受多种细胞因子调控， 转录因子结合顺式作用调控元件， HIV-1 LTR。这些细胞因子和病毒之间的相互作用 反式激活蛋白达特是基因调控的关键。除了 通过达特、多种其它调节剂调节HIV-1基因表达 信号转导通路的激活可以通过以下途径增加HIV-1基因的表达： 改变细胞转录因子的活性。是 上游转录因子的相互作用，一般的转录 机械，和达特，其导致HIV-1基因表达的激活。 这项拨款提案的重点是更好地了解复杂的 转录机制导致HIV-1复制增加。 特别是，我们希望进一步分析顺式作用调节 影响病毒复制的元素， 分级分离以鉴定对以下重要的细胞转录因子： HIV-1基因调控，并利用体外转录测定， 剖析达特转录途径的必要组成部分 activation.我们生产含有定点 每个关键的顺式作用HIV-1 LTR元件中的突变体， 固定化HIV-1模板测定的开发，其产生达特诱导的 增加HIV-1 LTR的体外转录， 新的和以前表征的一般和上游细胞 转录因子为我们提供了 为了更好地理解达特的激活。具体目标是 建议是：（l）使用与链霉亲和素结合的固定化HIV-1模板 珠来剖析转录途径中的步骤， 达特和细胞调节蛋白;（2）为了更好地表征 HIV-1中的特定序列和细胞因子 TATA元件影响基因表达的激活;（3）确定如何 RNA聚合酶II的转录延伸特性是 由细胞因子和达特修饰;（4）了解不同的 HIV-1 LTR调节区的突变改变病毒复制， 胸腺重建SCID-hu小鼠模型中的后续发病机制 以及（5）确定细胞的体内结合性质如何 HIV-1 LTR的因子在含有突变的病毒中发生改变， 达特基因或不同的HIV-1 LTR调控区。这些 研究将有助于解决细胞因子的作用， HIV-1基因表达。

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.