Regulation of Retroviral RNA Packaging and Dynamics of HIV-1 RNA Export

需要从细胞核中输出全长的HIV-1RNA来执行其功能，包括作为Gag/Gag-Pol翻译的模板，以及作为新组装的病毒粒子的基因组。为了研究HIV-1RNA包装，我们开发了一种新的方法，可以在单RNA分子敏感度下直接显示颗粒中的病毒RNA。这项检测显示，大多数HIV-1颗粒含有病毒RNA。尽管众所周知，包装成颗粒的逆转录病毒RNA是二聚体，但标准的生化分析不能确定一个颗粒中的RNA分子数量；我们的结果提供了证据，支持了长期存在的假设，即两个RNA分子(一个二聚体)被包装成一个颗粒。为了更好地了解RNA包装的机制，我们进行了遗传、生化和成像分析，表明HIV-1RNA利用DIS序列的碱基配对来选择其共包装的RNA伙伴，这一过程在RNA包装成颗粒之前发生在生产细胞的细胞质中。我们和其他人已经观察到，HIV-1RNA可以被操纵为偏离CRM1介导的核输出途径，而是通过大多数细胞mRNAs使用的Nxf1途径输出。然而，我们发现通过这两条途径输出的RNA分子并不能有效地复制，这表明输出途径影响了这些RNA的亚细胞定位。我们在这个项目中的未来努力将集中在使用高速、高分辨率显微镜来检测单个HIV-1RNA通过核孔复合体输出的动力学。我们将研究通过CRM1或NxF1途径的出口动力学，并探索不同的出口途径是否使用不同的核孔复合体。我们还将调查HIV-1用来调节包装了哪些和多少RNA的机制。此外，我们将利用RNA结合蛋白来重定向HIV-1RNA包装的特异性。这些研究将使我们了解对艾滋病毒复制至关重要的几个问题，这些问题可以用来制定阻止艾滋病毒传播的新战略。[对应于2011年10月艾滋病毒耐药性项目现场访问报告中的HU项目2]

Full-length HIV-1 RNA needs to be exported from the nucleus to carry out its functions including serving as a template for Gag/Gag-Pol translation and as a genome in the newly assembled virion. To study HIV-1 RNA packaging, we have developed a novel assay to directly visualize viral RNA in the particles at single-RNA-molecule sensitivity. This assay reveals that most HIV-1 particles contain viral RNA. Although it is known that retroviral RNAs packaged into particles are dimeric, standard biochemical assays cannot determine the number of RNA molecules in one particle; our results provide evidence to support the long-standing assumption that two RNA molecules (one dimer) are packaged into a particle. To better understand the mechanisms of RNA packaging, we have performed genetic, biochemical, and imaging analyses to show that HIV-1 RNA uses base-pairing of the DIS sequences to select its copackaged RNA partner, and this process occurs in the cytoplasm of the producer cell prior to RNA packaging into particles. We and others have observed that HIV-1 RNA can be manipulated to deviate from using the CRM1-mediated nuclear export pathway and, instead, be exported via the NXF1 pathway, which is used by most cellular mRNAs. However, we found that RNA molecules exported through these two pathways do not copackage efficiently, indicating that the export pathway affects subcellular localization of these RNAs. Our future efforts in this project will be focused on examining the dynamics of HIV-1 RNA export using high-speed, high-resolution microscopy to detect export of a single HIV-1 RNA through the nuclear pore complex. We will investigate the kinetics of export via the CRM1 or NXF1 pathways and explore whether different export pathways use different nuclear pore complexes. We will also investigate the mechanisms that HIV-1 uses to regulate which and how many RNAs are packaged. Additionally, we will utilize an RNA-binding protein to redirect HIV-1 RNA-packaging specificity. These studies will allow us to understand several questions that are fundamentally important to HIV replication, which can be used to generate new strategies to block the spread of HIV.[Corresponds to Hu Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]