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 该长末端重复序列作为细胞结合位点 转录因子。病毒反式激活蛋白Tat能够 通过刺激激活HIV-1 LTR的基因表达 转录延伸。在+1和+60之间的监管元素 能够形成稳定的茎环RNA的HIV-1 LTR 被指定为TAR的结构对TAT的激活至关重要。三要素 在包括上茎结构的TAR RNA中，有一个三核苷酸 凸起和六个碱基对的环对于TAT的激活都很重要。 Tat蛋白直接与TAR RNA凸起序列结合，最近 数据表明，结合TAR RNA区域的细胞因子，如 环路对于TAT功能可能很重要。这笔赠款的目标是 鉴定与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Tat in 结合结合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 具有转录起始或延长因子。 我们已经提纯了一种我们命名为Trp-185的细胞因子，并具有 最近克隆了编码这种因子的基因。Trp-185直接结合 与HIV-1的TAR RNA环序列的结合，尽管其结合取决于 一类被命名为Trp-185相关辅因子的细胞蛋白。 对Trp-185基因的分析表明，它编码一种新的蛋白质 它与原核Sigma因子有有趣的同源性。我们有 纯化了这些辅因子，并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结合蛋白中发现的基序，CF-2 与一组具有GTP酶活性的蛋白质具有同源性，以及 Cf-3与潜在的ATPase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的基础基因和TAT诱导的基因表达。这个 这项建议的具体目标是：(1)过度表达和纯化野生- 利用痘苗病毒表达TRP-185蛋白的类型和突变形式 载体，试图定义Trp-185的结构域，这些结构域涉及 TAR RNA结合和转录激活；(2)测定 辅因子蛋白促进Trp-185的机制 与TAR RNA结合，并确定TAT是否修改了这一过程； 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或 转录激活；以及(4)使用体外转录检测 用纯化的RNA聚合酶II和通用转录因子 确定Trp-185和Tat的潜在细胞靶点。这些研究 对于理解细胞因子的功能将是重要的 与TAR RNA结合并确定它们在调节TAT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.