HIV Molecular Biology and Pathogenic Mechanisms of AIDS

摘要我们利用突变的HIV-1DNA前病毒克隆作为一种新的分子陷阱，在小鼠基因组中发现了一个广泛的RNA转运元件(RTE)家族，能够取代HIV-1Rev/RRE转录后调控系统。这是鉴定哺乳动物基因组中顺式作用转录后调控元件的一般方法。我们已经详细分析了RTE元件的结构和功能，并确定了负责与RTE结合并将其与Nxf1输出途径联系起来的细胞因子。这种蛋白质是RNA结合基序蛋白15(RBM15)，以前没有指定的功能。RBM15在体外直接和特异性地识别RTE，并在体内刺激含有RTE的报告mRNAs的输出和表达。将RBM15与报告基因mRNA连接，结果表明，RBM15通过促进核内的mRNA输出发挥作用。我们还发现，RBM15与Nxf1结合，这两种蛋白在刺激RTE介导的mRNA输出和表达方面存在协同作用。因此，RBM15是一个新的信使核糖核酸输出因子，是Nxf1途径的一部分。我们认为，RTE进化为一个高亲和力的RBM15配体，提供与Nxf1的剪接无关的连接，从而确保高效的核输出和逆转录转座子转录产物的表达。与这些数据一致，Barbara Felber的研究小组现在已经表明，全长、活跃的逆转座子中的RTE位点对于逆转座子是必不可少的。我们的结果有助于进一步理解大分子核质运输的基本机制。了解HIV基因表达的调控机制具有重要的实际应用价值。我们已经将这一知识应用于改进的DNA疫苗接种方法的开发。我们还表明，CTE和RTE元件在同一个mRNA中的组合可以用来增加基因表达一个数量级以上。我们已经利用先前开发的RNA优化技术来优化IL-15细胞因子的表达，并证明了我们可以在小鼠和猕猴体内传递DNA后产生生物活性细胞因子。我们构建了新的逆转录病毒载体，当插入到基因区域时，可以有效地终止转录/翻译，并构建了小鼠胚胎干细胞的有序文库，可以用来生产出任何想要的敲除小鼠。这项技术简化了KO小鼠的生产和研究，并有助于快速了解基因功能。该方法通过为57个GPCR基因和几个核受体基因开发小鼠KO来验证，其中大多数基因在文献中不存在。我们利用优化的表达载体在动物体内表达IL-12或IL-15细胞因子。有效的表达导致这些细胞因子的生物活性水平。我们正在优化这些强大的细胞因子的剂量，使它们成为我们疫苗的重要分子佐剂。我们已经证明，生物活性IL-15的主要形式(如果不是唯一的形式)与其自身的IL-15受体α形成复合体。我们还发现，在DNA疫苗接种后，IL-12是一种强大的免疫反应诱导剂。

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.