THE REV/REX TRANSACTIVATION PATHWAY OF HIV/HTLV

描述：（改编自申请人摘要）申请人已 观察到约56 kD的单个核因子（NF-RRE）结合 到RRE的近端区域。 此外，两个Rev 和NF-RRE导致形成包含两种蛋白质的单一复合物 的RRE。 申请人的主要目标是研究结构， NF-RRE的功能及其在Rev介导的反式激活中的作用， 转录后基因调控 此外，研究者将 试图确定和描述类似的核因素， 参与其他人类逆转录病毒的类似途径。 第一 具体目标是试图查明核因素并确定其特征 结合HIV/RRE和人T细胞嗜淋巴细胞病毒（HTLV）/RxRE 序列的 研究者将尝试确定是否相同或不同 核因子识别这些不同的RNA靶序列。 的 申请人建议对Rev RRE和RxRE上的结合位点进行精细定位 和核因子，使用诱变以及指纹， 足迹技术 Wong-Staal博士的第二个目标是尝试 部分纯化NF-RRE蛋白并制备单特异性和 单克隆抗体。这些试剂将用于初始 蛋白质的功能表征并可能产生探针 用于NF-RRE基因的克隆。 接下来，申请人提出克隆和 表达NF-RRE基因。 这将尝试使用几个替代方案 实现基因克隆的策略，取决于是否辅助 蛋白质是NF-RRE-RRE结合所必需的。 一旦获得，基因 将在原核载体中表达，以获得大量的 纯化的蛋白质并在真核载体中用于功能分析。 的 研究者的最后一个目标是NF-RRE的功能表征。 核苷酸序列测定、免疫耗竭和重建 使用体外剪接和剪接体组装测定的研究， 反义或反式显性突变构建体在哺乳动物中的表达 细胞将进行。 这些将被做尝试确定 NF-RRE结构、功能及其在mRNA剪接/转运中的作用 和Rev介导的反式激活。

DESCRIPTION: (Adapted from applicant's abstract) The applicant has observed that a single nuclear factor (NF-RRE) of approximately 56 kD binds to the proximal region on the RRE. Furthermore, the presence of both Rev and NF-RRE result in formation of a single complex comprising both proteins and RRE. The major goal of the applicant is to study the structure and function of NF-RRE and its role in Rev-mediated transactivation as well as post-transcriptional gene regulation. In addition, the investigator will attempt to identify and characterize similar nuclear factors that are involved in analogous pathways of other human retroviruses. The first specific aim is to attempt to identify and characterize nuclear factors that bind HIV/RRE and human T cell lymphotropic virus (HTLV)/RxRE sequences. The investigator will try to determine if the same or different nuclear factors recognize these various RNA target sequences. The applicant proposes to fine map the binding sites on RRE and RxRE for Rev and nuclear factor(s) using mutagenesis as well as fingerprinting and footprinting techniques. The second goal of Dr. Wong-Staal is to attempt a partial purification of NF-RRE protein and to prepare monospecific and monoclonal antibodies to it. These reagents will be used in initial functional characterization of the protein and possibly to generate probes for cloning of the NF-RRE gene. Next, the applicant proposes to clone and express the NF-RRE gene. This will be attempted using several alternative strategies to achieve cloning of the gene, depending on whether accessory protein(s) are required for NF-RRE-RRE binding. Once obtained, the gene will be expressed in prokaryotic vectors to obtain large amounts of purified protein and in eukaryotic vectors for functional analyses. The last goal of the investigator is the functional characterization of NF-RRE. Nucleotide sequence determination, immunodepletion and reconstitution studies using in vitro splicing and spliceosome assembly assays and expression of anti-sense or transdominant mutant constructs in mammalian cells will be carried out. These will be done to try to determine the structure and function of NF-RRE and its role in mRNA splicing/transport and Rev-mediated transactivation.