REV/REX TRANSACTIVATION PATHWAY OF HIV/HTLV

描述：(改编自申请人的摘要)申请人有 观察到大约56kD的单个核因子(NF-RRE)结合 在RRE上的近端区域。此外，两个Rev的存在 和NF-RRE导致形成由这两种蛋白质组成的单一复合体 和RRE。申请者的主要目标是研究结构和 核因子-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，调查员将 试图识别和描述类似的核因素，这些因素 参与了其他人类逆转录病毒的类似途径。第一 具体目标是试图识别和表征核因素 结合HIV/RRE和人类T淋巴细胞嗜淋巴病毒(HTLV)/RxRE 序列。调查员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。这个 申请人建议精细绘制REV的RRE和RxRE上的结合位点图 和核因子(S)采用诱变以及指纹图谱和 足迹技术。黄之锋博士的第二个目标是尝试 部分纯化核因子-RRE蛋白并制备单特异性和 抗它的单抗。这些试剂将在初期使用 蛋白质的功能特性，并可能产生探针 用于克隆核因子-RRE基因。下一步，申请人提议克隆和 表达核因子-RRE基因。我们将尝试使用以下几种替代方案 实现基因克隆的策略，取决于是否有附属 蛋白质(S)是NF-RRE-RRE结合所必需的。一旦获得，该基因 将在原核载体中表达，以获得大量的 纯化的蛋白和用于功能分析的真核载体。这个 研究人员的最后目标是对核因子-RRE的功能进行表征。 核苷酸序列测定、免疫耗竭和重组 体外剪接和剪接体组装试验的研究 反义或跨显性突变结构在哺乳动物中的表达 细胞将会被执行。将执行这些操作，以尝试确定 核因子-RRE的结构和功能及其在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.