REGULATION OF HIV RNA PROCESSING AND TRANSPORT

人类免疫缺陷病毒(HIV)rev蛋白功能促进 未剪接和未完全剪接的艾滋病毒mRNAs的核出口。Rev可能会 也在这些mRNAs的翻译中发挥作用。我们的最新结果 实验室研究表明，REV与细胞中含有内含子的HIV-RNA结合 在也包含剪接因子的复合体中。更多数据表明 该REV直接与参与剪接的SnRNP蛋白结合。这些 结果支持这样的概念，即rev与 剪接机械解决剪接因数受转速调节 RNA。 这是一项修订后的提案，描述了旨在进一步 阐明rev在RNA加工和运输中的作用。为了做到这一点，我们 将表征REV和RRE之间形成的络合物 转基因细胞和体外剪接提取液中的RNA。这些 实验将包括对细胞蛋白质和RNA的分析 这些复合体。我们将进一步分析rev和 在没有含RRE的RNA的情况下，细胞蛋白质和RNA。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是细胞核和细胞质之间的穿梭蛋白， 多聚体复合体中是否存在REV。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最近，我们 已经在梅森辉瑞猴子病毒的3‘端发现了一个元件 在HIV表达中替代REV和RRE的(MPMV)基因组 复制。我们假设这个元素直接与 影响内含子转运的构成细胞因子 核糖核酸。MPMV元素将进一步使用随机性和位置- 特定的突变作用。此外，我们将尝试识别细胞 结合在这种元素上的蛋白质。这些蛋白质的基因将被克隆 并对蛋白质的性质进行了分析。最后，我们会 分析感染细胞的RNA加工和结构蛋白表达 含有MPMV成分的病毒。 本研究的长期目标是进一步阐明心力衰竭的机制。 HIV rev蛋白的作用。进一步了解这种蛋白质是如何 促进非剪接RNA的表达，可能使新颖的设计成为可能 转速功能的抑制剂。因为版本对病毒来说是绝对必要 复制，这可能导致有效抗击艾滋病的新药。

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.