This proposal focuses on the practical development of nucleic acid pharmaceuticals that can be used for the treatment of AIDS. Regulation of HIV replication is dependent on a programmed series of protein:nucleic acid interactions. In particular, the regulatory factor Rev may control HIV-1 latency and viral reactivation. Rev facilitates the cytoplasmic transport of mRNAs encoding viral structural proteins by binding to a cis- acting Rev-responsive element (RRE). Disruption of the interaction between Rev and the RRE has been shown to block the timely production of structural proteins and effectively disrupts the HIV life cycle. An in vitro genetic selection has been used to isolate novel nucleic acid 'decoys' that can bind the Rev protein an order of magnitude better than the wild-type RRE. Additional selections are designed to isolate decoys with further enhanced affinities. In order to assess whether the selected decoys can serve as anti-HIV pharmaceuticals their ability to inhibit Rev:RRE interactions and HIV replication in tissue culture cells will be assayed. Experiments that seek to establish the dose-response relationship for inhibition have also been included; these experiments should prove useful in designing gene therapies. Sequence analysis of the selected Rev decoys has led to the development of a structural model for interactions between Rev and the RRE. This model will be tested and refined by physical mapping studies, and by in vitro selection of sequence covariations in both peptide and nucleic acid ligands. The model represents a wealth of structural details that can be used to design chemical mimetics of the RRE that can potentially be used as drugs for the treatment of AIDS. In particular, the peptide motifs identified by physical mapping and selection studies should provide information essential to the synthesis of peptidomimetic drugs.
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