REGULATION OF HIV RNA PROCESSING AND TRANSPORT

人类免疫缺陷病毒（HIV）rev蛋白的功能是促进 未剪接和不完全剪接的HIV mRNA的核输出。Rev May 也在这些mRNA的翻译中发挥作用。最近的结果在我们的 实验室表明，rev与细胞中含有内含子HIV RNA结合 在一个含有剪接因子的复合体中。其他数据显示， rev直接与参与剪接的snRNP蛋白结合。这些 结果支持rev与 从转速调节上解决拼接机械的拼接因素 RNA。 这是一份修订后的提案，描述了旨在进一步 阐明rev在RNA加工和转运中的作用。为此我们 将表征rev和含RRE的 转染细胞和体外剪接提取物中的RNA。这些 实验将包括对细胞蛋白质和RNA的分析 这些复合物。我们将进一步分析rev和 细胞蛋白质和RNA在不存在含RRE的RNA的情况下。这些 实验将确定rev是否参与了 新型snRNP颗粒。在这些研究中，我们还将调查 rev作为细胞核和细胞质之间的穿梭蛋白， rev是否存在于多核糖体复合物中。一种新的酵母系统将是 用于识别与Rev相互作用的细胞蛋白质。最近，我们 已经在梅森辉瑞猴病毒的3'端发现了一个元素 在HIV表达中替代rev和RRE的MPMV基因组， 复制的我们假设这种元素直接与 组成性细胞因子，以实现含内含子的转运 核糖核酸MPMV元素将进一步使用随机和现场表征。 特异性诱变此外，我们将尝试识别细胞 蛋白质与该元素结合。这些蛋白质的基因将被克隆 并分析蛋白质的性质。最后我们将 分析感染细胞中的RNA加工和结构蛋白表达 含有MPMV元件的病毒。 本研究的长期目标是进一步阐明 HIV rev蛋白的作用。进一步了解这种蛋白质 促进未剪接RNA的表达，可能使设计新的 rev功能的抑制剂。因为rev对病毒是绝对必要的 复制，这可能导致新的药物，以有效地打击艾滋病。

The Human Immunodeficiency Virus (HIV) rev protein functions to facilitate nuclear export of unspliced and incompletely spliced HIV mRNAs. Rev may also play a role in translation of these mRNAs. Recent results in our laboratory indicate that rev binds to intron-containing HIV-RNA in cells in a complex that also contains splicing factors. Additional data suggest that rev binds directly to snRNP proteins involved in splicing. These results support the notion that rev interacts with components of the splicing machinery to resolve splicing factors from the rev regulated RNAs. This is a revised proposal that describes experiments designed to further clarify the role of rev in RNA processing and transport. To do this , we will characterize the complexes that form between rev and RRE-containing RNA in transfected cells and in in vitro splicing extracts. These experiments will include an analysis of the cellular proteins and RNAs in these complexes. We will further analyze interactions between rev and cellular proteins and RNA in the absence of RRE-containing RNA. These experiments will determine whether rev participates in the formation of a novel snRNP particle. In these studies we will also investigate whether rev acts as a shuttle protein between the nucleus and the cytoplasm and whether rev is present in polysome complexes. A novel yeast system will be used to identify cellular proteins that interact with rev. Recently, we have identified an element at the 3' end of the Mason Pfizer Monkey Virus (MPMV) genome that substitutes for rev and the RRE in HIV expression and replication. We hypothesize that this element interacts directly with constitutive cellular factors to effectuate transport of intron containing RNA. The MPMV element will be further characterized using random and site- specific mutagenesis. In addition, we will attempt to identify cellular proteins binding to this element. Genes for such proteins will be cloned and the properties of the proteins will be analyzed. Finally, we will analyze RNA processing and structural protein expression in cells infected with virus containing the MPMV element. The long term goal of this research is to further clarify the mechanism of action of the HIV rev protein. Further insight into how this protein promotes the expression of unspliced RNA, might enable the design of novel inhibitors of rev function. Since rev is absolutely essential for virus replication, this could lead to new drugs to efficiently combat AIDS.