CELLULAR PROTEINS INVOLVED IN HIV 1 TAR RNA FUNCTION

HIV-1基因表达依赖于不同的调控元件， 作为细胞结合位点的长末端重复序列 转录因子 病毒反式激活蛋白达特能够 激活HIV-1 LTR的基因表达， 转录延伸 在+1和+60之间的调节元件， 能够形成稳定茎环RNA的HIV-1 LTR 命名为TAR的结构对于达特活化是关键的。 三个要素 在TAR RNA中，包括上部茎结构， 凸起和六个碱基对环对于达特激活都是重要的。 达特蛋白直接结合到TAR RNA凸起序列，并且最近 数据表明，与TAR RNA区域结合的细胞因子， 该环路对于达特功能可能是重要的。 该补助金的目标是 为了表征与HIV-1结合的细胞因子的功能， TAR RNA环序列。 我们希望测试的模型是达特 结合TAR RNA的细胞因子能够 通过直接或间接相互作用激活HIV-1基因表达 与转录起始因子或延伸因子结合。 我们纯化了一种细胞因子，命名为TRP-185， 最近克隆了编码这种因子的基因。 TRP-185直接结合 HIV-1 TAR RNA环序列，尽管其结合依赖于 一类称为TRP-185相关辅因子的细胞蛋白质。 对TRP-185基因的分析表明，它编码一种新的蛋白质 其与原核σ因子具有有趣的同源性。 我们有 并对这些辅因子进行了纯化，克隆了三个辅因子的基因 命名为CF-1、CF-2和CF-3的蛋白质。 CF-1基因包含一个 类似于在各种RNA结合蛋白CF-2中发现的基序 与一组具有GTPase活性的蛋白质具有同源性，并且 CF-3与潜在的ATP酶或蛋白激酶具有同源性。 现在 我们有编码这些基因的cDNA克隆，我们将研究它们在 介导HIV-1 LTR的基础和tat诱导的基因表达。 的 本发明的具体目的是：（1）过量表达和纯化野生型- 使用牛痘表达的TRP-185蛋白的类型和突变形式 载体，试图定义TRP-185的结构域， TAR RNA结合和转录激活;（2）为了确定TAR RNA结合和转录激活的可能性， 辅助因子蛋白能够促进TRP-185的机制 结合TAR RNA，并确定达特是否修饰该过程;（3） 鉴定与TRP-185直接相互作用的细胞蛋白， 确定这些蛋白质是否影响其结合特性， 转录激活;和（4）使用体外转录测定 用纯化的RNA聚合酶II和一般转录因子， 鉴定TRP-185和达特的潜在细胞靶点。 这些研究 对理解细胞因子的功能很重要， 与TAR RNA结合并确定它们在调节达特功能中的作用。

HIV-1 gene expression is dependent on distinct regulatory elements in the long terminal repeat that serve as binding sites for cellular transcription factors. The viral transactivator protein tat is able to activate gene expression from the HIV-1 LTR by stimulating transcriptional elongation. A regulatory element between +1 and +60 in the HIV-1 LTR which is capable of forming a stable stem-loop RNA structure designated TAR is critical for tat activation. Three elements within TAR RNA including the upper stem structure, a three nucleotide bulge, and a six base pair loop are all important for tat activation. Tat protein directly binds to the TAR RNA bulge sequences, and recent data suggests that cellular factors binding to TAR RNA regions such as the loop may be important for tat function. The goal of this grant is to characterize the function of cellular factors that bind to the HIV-1 TAR RNA loop sequences. The model that we wish to test is that tat in conjunction with cellular factors binding to TAR RNA are able to activate HIV-1 gene expression by either direct or indirect interactions with transcription initiation or elongation factors. We have purified a cellular factor that we designated TRP-185 and have recently cloned the gene encoding this factor. TRP-185 binds directly to the HIV-1 TAR RNA loop sequences though its binding is dependent on a class of cellular proteins designated TRP-185 associated cofactors. Analysis of the TRP-185 gene indicates that it encodes a novel protein which has interesting homology with procaryotic sigma factors. We have also purified these cofactors and cloned the genes of three cofactor proteins designated CF-1, CF-2, and CF-3. The CF-1 gene contains a motif similar to those found in a variety of RNA binding proteins, CF-2 has homology with a group of proteins which have GTPase activity, and CF-3 has homology with potential ATPases or protein kinases. Now that we have cDNA clones encoding these genes, we will study their role in mediating basal and tat-induced gene expression of the HIV-1 LTR. The specific aims of this proposal are: (1) To overexpress and purify wild- type and mutated forms of the TRP-185 protein using vaccinia expression vectors in an attempt to define domains of TRP-185 that are involved in TAR RNA binding and transcriptional activation; (2) To determine the mechanism by which cofactor proteins are able to facilitate TRP-185 binding to TAR RNA and to determine if tat modifies this process; (3) To identify cellular proteins that directly interact with TRP-185 and determine if these proteins influence either its binding properties or transcriptional activation; and (4) To use in vitro transcription assays with purified RNA polymerase II and general transcription factors to identify potential cellular targets for TRP-185 and tat. These studies will be important in understanding the function of cellular factors that bind to TAR RNA and determining their role in modulating tat function.