HIV Molecular Biology and Pathogenic Mechanisms of AIDS

我们以前已经鉴定了一个广泛的RNA转运元件家族 (RTE)在小鼠基因组中能够取代HIV-1 Rev/RRE转录后调节 系统，使用突变的HIV-1 DNA前病毒克隆作为一种新的分子陷阱。这是将军 方法鉴定顺式作用转录后控制元件在 哺乳动物基因组详细分析了RTE元件的结构和功能， 我们已经确定了负责与RTE结合的细胞因子，并将其与 NXF 1输出途径。这种蛋白质，即RNA结合基序蛋白15（RBM 15）， 以前分配的功能。RBM 15在体外直接特异性识别RTE， 在体内刺激含有RTE的报告基因mRNA的输出和表达。RBM 15的系留 对报告基因mRNA的研究表明，RBM 15通过促进mRNA从细胞核输出而起作用。我们也 发现RBM 15与NXF 1结合，这两种蛋白质在刺激RT介导的mRNA中协同作用 出口和表达。因此，RBM 15是一种新的mRNA输出因子，是NXF 1的一部分， 通路我们认为RTE进化为高亲和力RBM 15配体， 与NXF 1的非剪接依赖性连接，从而确保有效的核输出和表达， 反转录转座子转录本。与这些数据一致，芭芭拉·费尔伯的研究小组现在已经 表明全长内的RTE位点，活性反转录转座子是必不可少的 反转录转位我们的研究结果有助于进一步了解 大分子核质运输的基本机制。的理解 HIV基因表达调控机制的研究具有重要的实际应用价值。我们 已经将这些知识应用于改进的DNA疫苗接种方法的开发。我们也 结果表明，相同mRNA中CTE和RTE元件的组合可用于增加 基因的表达量增加了不止一个数量级我们使用了以前开发的 RNA优化技术来优化IL-15细胞因子的表达，并且已经显示， 在小鼠和猕猴中，我们可以在DNA递送后产生具有生物活性的细胞因子。我们已经构建 新的逆转录病毒载体，其在插入时有效终止转录/翻译， 基因区域，并已构建了有序的小鼠胚胎干细胞库， 用于产生具有任何所需敲除的小鼠。这项技术简化了生产 和KO小鼠的研究，可以帮助快速了解基因功能。述的方法 通过开发小鼠科斯的57个GPCR基因和几个核受体基因， 其中大部分在文献中是不存在的。我们使用优化的表达载体， 在动物中表达IL-12或IL-15细胞因子。高效表达导致生物活性水平 这些细胞因子。我们正在优化这些强大的细胞因子的剂量， 成为疫苗的重要分子佐剂。我们已经证明，如果不是， 生物活性IL-15的唯一形式是与其自身的IL-15受体α复合。我们有 也表明IL-12是DNA疫苗接种后免疫应答的强有力诱导剂。

Summary We have previously identified an extensive family of RNA transport elements (RTE) in the mouse genome able to replace the HIV-1 Rev/RRE posttranscriptional regulatory system, using a mutated HIV-1 DNA proviral clone as a novel molecular trap. This is general methodology for the identification of cis-acting posttranscriptional control elements in the mammalian genome. We have analyzed in detail the structure and function of the RTE element, and we have identified the cellular factor responsible for binding to RTE and linking it to the NXF1 export pathway. This protein, the RNA binding motif protein 15 (RBM15), had no previous assigned function. RBM15 recognizes RTE directly and specifically in vitro, and stimulates export and expression of RTE-containing reporter mRNAs in vivo. Tethering of RBM15 to a reporter mRNA showed that RBM15 acts by promoting mRNA export from the nucleus. We also found that RBM15 binds to NXF1 and the two proteins cooperate in stimulating RTE-mediated mRNA export and expression. Thus, RBM15 is a novel mRNA export factor and is part of the NXF1 pathway. We propose that RTE evolved as a high-affinity RBM15 ligand to provide a splicing-independent link to NXF1, thereby ensuring efficient nuclear export and expression of retrotransposon transcripts. Consistent with these data, the group of Barbara Felber has now shown that the RTE site within full-length, active retrotransposons is essential for retrotransposition. Our results contribute significantly to the further understanding of the basic mechanisms of nucleocytoplasmic traffic of macromolecules. The understanding of the regulatory mechanisms of HIV gene expression has many important practical applications. We have applied this knowledge to the development of improved DNA vaccination approaches. We also showed that the combination of CTE and RTE elements in the same mRNA can be used to increase gene expression by more than one order of magnitude. We have used the previously developed technologies of RNA optimization to optimize expression of IL-15 cytokine, and have shown that we can produce bioactive cytokine after DNA delivery in mice and macaques. We have constructed new retroviral vectors that efficiently terminate transcription/translation when inserted in gene regions, and have constructed ordered libraries of mouse embryonic stem cells that can be used to produce mice having any desired knock out. This technology streamlines the production and study of KO mice and can assist in the rapid understanding of gene function. The method was validated by developing mouse KOs for 57 GPCR genes and several nuclear receptor genes, most of which did not exist in the literature. We have used optimized expression vectors to express IL-12 or IL-15 cytokines in animals. Efficient expression results in bioactive levels of these cytokines. We are optimizing the dose of these powerful cytokines so that they will become an important molecular adjuvant for our vaccines. We have shown that the major, if not the only one, form of bioactive IL-15 is in complex with its own IL-15 Receptor alpha. We have also shown that IL-12 is a powerful inducer of immune responses after DNA vaccination.