The genome of HIV codes for two proteins, Tat and Rev, which are essential regulators of transcription. Rev binds to a specific RNA sequence called the Rev Responsive Element (RRE), which is found in the HIV genome and regulates the cytoplasmic appearance of unspliced or singly spliced mRNAs which encode the structural proteins gag, pol, and env3. Rev has been proposed to have a role both in regulation of splicing and in transport of the RNA to the cytoplasm. Chemical and RNAse protection experiments have shown that a 66 nucleotide fragment, domain II of the RRE, is necessary and sufficient for high affinity binding of Rev in vitro and that domain II alone is sufficient for a detectable Rev responsiveness in vivo. The core Rev binding element within the RRE and the critical bases or base-base interactions have recently been more precisely defined by an iterative in vitro genetic selection as well as other techniques. RNA oligonucleotides 30-35 nucleotides long containing the core binding element were shown to bind Rev with wild type affinity. Other studies have shown that a 17 amino acid arginine rich peptide from Rev binds RRE at multiple sites and specifically inhibits splicing in vitro. A single peptide specifically binds in the core elements of the RRE. We propose to use multidimensional, multinuclear NMR spectroscopy to elucidate the structural features of the RRE that are important in Rev recognition and binding. The specific structural studies which will be undertaken are: 1. Determination of the three-dimensional structures of several RNA oligonucleotides 30-35 bases in length containing the core binding element of RRE for Rev. These will include both wild-type sequences, sequence variants which contain compensatory double mutations, non-functional variants, and variants with binding affinities that are greater than the wild type. We hope to extend these studies to the entire 66 nucleotide domain II region of RRE. 2. Determination of the three-dimensional structure of complexes formed between Rev peptides and the core binding element of RRE. Various RNA sequences complexed with the 17 amino acid Rev peptide will be studied. A longer range goal is to solve the structure of Rev complexed to a short RRE oligonucleotide. An understanding of how Rev binds to its RNA may help in the development of strategies designed to block viral spread through disruption of essential protein activities. In addition, these studies should lead to a better general understanding of the determinants for protein binding to RNA as well as the rules governing folding and tertiary interactions in RNA. In addition to the RRE-Rev projects outlined above, we also plan to collaborate with Professors Eisenberg, Dickerson, and Sigman on three- dimensional structure determination of relevant proteins, peptides, RNAs, complexes of these and drug-RNA:DNA complexes.
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