Background: HIV and HIV-related proteins are produced by recombinant DNA methods for high-resolution structural analyses. The proteins are selected which are important for the life cycle of the virus and for its structural integrity and, thus, represent potential targets for rational structure-based drug design. Collaborating groups performed the actual structure determinations. The determination of the 3-D structure of proteins by X-ray diffraction or multidimensional NMR required the production of large quantities of highly purified and physically homogeneous protein. In addition, protein structure determination by NMR requires protein biosynthetically labeled with combinations of the stable isotopes: H-2, C-13 and N-15. Results: (1) HIV Rev is an important regulatory factor required for HIV expression. We have made extensive modifications of the Rev protein in order to improve its solubility for NMR measurements and crystallization trials. In addition, we are attempting to further stabilize Rev for structural studies by forming binary complexes with antibodies and tubulin. To generate a wider range of monoclonal antibodies for co-crystallization studies, we are making recombinant antibodies based on phage display selection. (2) Nef is a 23 kDa protein essential for the pathogenic properties of HIV. We are continuing to investigate some of the specific protein-protein interactions involving Nef especially involving the HIV-1 transactivating protein Tat. We have used surface plasmon resonance to show a specific and tight binding between these proteins. Using protein engineered variants of Nef and Tat we are preparing complexes for more detailed structural study (3) HIV protease, a homodimeric protein is essential in the viral life cycle and a major anti-AIDS drug target. Peptides derived from the N- and C-terminal regions of the HIV-1 protease dimer interface, inhibit protease by preventing dimerization. The solubility and cell permeability of the peptides was enhanced by linking the transduction domain of HIV-1 Tat. Summary: the immunodeficiency virus (HIV) comprises a number of proteins with regulatory and structural roles. HIV proteins important for the virus life cycle, and proteins which have anti-HIV activity, are expressed in bacteria using recombinant DNA methods. The proteins are purified then studied to establish their chemical and physical properties. Well-characterized proteins are made available to NIH investigators who study the molecular structure of these proteins. This structural information may provide impetus for targeted drug design and discovery.
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