Packaging Signal Interactions in HIV & Other RNA Viruses

描述（由申请人提供）：我们研究的广泛长期目标是表征逆转录病毒中包装基因组RNA的分子相互作用。这一认识为设计和评价干扰该过程的药物提供了基础。在我们成功地首次精确测量了参与包装的RNA和蛋白质组分的亲和力，并通过NMR确定了重要组分的3D结构之后，我们的总体目标是：(A)分析HIV-1和MMTV RNA-核衣壳（RNA- nc）复合物中相互作用的稳定性和多样性。(B)通过NMR确定必需RNA和RNA- nc复合物的高分辨率结构。在这个项目期间，我们将使用核磁共振和荧光光谱来表征HIV-1和MMTV的5'-leader的特定RNA亚结构，以及它们与逆转录病毒蛋白的核衣壳结合域的相互作用。计划的主要生物靶标是参与包装过程的5'-先导序列的亚结构。该项目的分析和信息应该有助于设计和筛选候选药物，这些候选药物可以开发为包装抑制剂。具体来说，我们的目标是(1)将我们的NCp7色氨酸荧光法应用于野生型和变异的HIV-1茎环，以确定相互作用的位点。(2)开发一种快速、灵敏的检测方法，用于评估紧密结合RNA-NC复合物在NCp7和gag前体蛋白中的亲和力。(3)探讨RNA-NC在MMTV中的相互作用。(4)获得游离RNA和RNA- nc复合物的高分辨率结构，其中RNA来源于具有nc结合活性的5'-前导区。

DESCRIPTION (provided by applicant): The broad long-term objective of our research is to characterize the molecular interactions responsible for packaging genomic RNA in retroviruses. This understanding provides a basis for the design and evaluation of pharmaceuticals to interfere with the process. Following our success in achieving the first accurate measurements of the affinities of RNA and protein components involved in packaging, and in determining 3D structures of important components by NMR, our general aims are to: (A) Analyze the stability and diversity of interaction in HIV-1 and MMTV RNA-nucleocapsid (RNA-NC) complexes. (B) Determine high-resolution structures of essential RNA and RNA-NC complexes by NMR. During this project period, we will use NMR, and fluorescence spectroscopy to characterize specific RNA sub-structures from the 5'-leaders of HIV-1 and MMTV, and their interactions with the nucleocapsid binding domain of retroviral proteins. The primary biological targets planned are substructures from the 5'-leader sequence involved in the packaging process. The assay and information from the project should be useful in designing and screening drug candidates that could be developed as packaging inhibitors. Specifically, we aim to (1) Apply our NCp7 tryptophan fluorescence assay to wild-type and variant stemloops of HIV-1 to determine the loci of interaction. (2) Develop a rapid and sensitive assay that will be useful for assessing the affinities of tight-binding RNA-NC complexes, both in NCp7 and in gag-precursor proteins. (3) Explore RNA-NC interactions in MMTV. (4) Obtain high-resolution structures of free RNA and RNA-NC complexes, where the RNA is derived from the 5'-leader regions that exhibit NC-binding activity.