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.
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