HIV Molecular Biology and Pathogenic Mechanisms of AIDS

HIV分子生物学与艾滋病发病机制

• 批准号: 7733193
• 负责人: George N. Pavlakis
• 金额: $ 44.19万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733193
• 关键词: Acquired Immunodeficiency Syndrome; Adjuvant; Affinity; Amino Acid Motifs; Animals; Basic Cancer Research; Binding; Binding Sites; Biochemical Genetics; Cell Nucleus; Cell physiology; Cells; Complementary DNA; Complex; DNA; DNA Vaccines; DNA delivery; Data; Development; Disease; Dose; Doxycycline; Elements; Ensure; Evolution; Family; G Protein-Coupled Receptor Genes; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; HIV; HIV-1; Immune response; Immunotherapy; In Vitro; Indium; Interleukin-12; Interleukin-15; Intervention; Knock-out; Knockout Mice; Knowledge; Length; Libraries; Ligands; Link; Literature; Macaca; Malignant Neoplasms; Mediating; Messenger RNA; Methodology; Methods; Molecular; Molecular Biology; Mus; Mutagenesis; Mutate; Names; Nuclear Export; Nuclear Receptor Gene; Pathologic Processes; Pathway interactions; Process; Production; Proteins; RNA; RNA Binding; RNA Splicing; RNA Transport; RNA-Binding Proteins; Reporter; Retrotransposition; Retrotransposon; Retroviral Vector; Retroviridae; Site; Structure; Subfamily lentivirinae; System; Technology; Tetracycline; Tetracyclines; Trans-Activators; Transcript; Translations; Vaccination; Vaccines; cytokine; desire; embryonic stem cell; expression vector; gene function; gene therapy; gene transfer vector; improved; in vivo; interleukin-15 receptor; knockout gene; mRNA Export; macromolecule; mammalian genome; mouse genome; new technology; novel; practical application; prevent; recombinase; tool; trafficking; vector

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.

综上所述，我们先前使用突变的HIV-1 DNA病毒前克隆作为一种新型分子陷阱，以前已经确定了小鼠基因组中广泛的RNA转运元件（RTE）家族。这是鉴定哺乳动物基因组中的顺式作用后控制元件的一般方法。我们已经详细分析了RTE元素的结构和功能，并确定了负责与RTE结合并将其链接到NXF1导出途径的细胞因子。该蛋白是RNA结合基序蛋白15（RBM15），没有以前的分配函数。 RBM15直接识别RTE，并在体外识别RTE，并刺激体内含有RTE的记者mRNA的出口和表达。将RBM15绑定到报告基因mRNA上表明，RBM15通过促进从细胞核出口的mRNA导出来起作用。我们还发现，RBM15与NXF1结合，两种蛋白在刺激RTE介导的mRNA输出和表达方面合作。因此，RBM15是一种新型的mRNA输出因子，是NXF1途径的一部分。我们建议RTE演变为高亲和力RBM15配体，以提供与NXF1的剪接无关的链接，从而确保了逆转录跨跨座子转录本的有效核输出和表达。与这些数据一致，芭芭拉·费尔伯（Barbara Felber）组现已表明，在全长，主动返回转座子中的RTE位点对于逆转录座是必不可少的。我们的结果显着有助于进一步理解大分子核细胞质流量的基本机制。对HIV基因表达的调节机制的理解具有许多重要的实际应用。我们已经将这些知识应用于改进的DNA疫苗接种方法的发展。我们还表明，同一mRNA中的CTE和RTE元素的组合可用于将基因表达提高多个数量级。我们已经使用了先前开发的RNA优化技术来优化IL-15细胞因子的表达，并表明我们可以在小鼠和猕猴中DNA后产生生物活性细胞因子。我们已经构建了新的逆转录病毒载体，这些向量在基因区域插入时有效终止转录/翻译，并构建了小鼠胚胎干细胞的有序文库，这些库可用于产生任何所需的敲除。该技术简化了KO小鼠的生产和研究，可以帮助快速理解基因功能。该方法通过为57个GPCR基因和几个核受体基因开发小鼠KOS来验证，其中大多数在文献中不存在。我们已经使用了优化的表达矢量来表达动物中的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，以便它们成为我们疫苗的重要分子佐剂。我们已经表明，生物活性IL-15的主要形式（即使不是仅有的）形式与其自身的IL-15受体α相当复杂。我们还表明，IL-12是DNA疫苗接种后免疫反应的强大诱导剂。