(1) The HIV protein Rev is an essential for viral replication controlling expression of regulatory and structural proteins. The structure of Rev has not been determined due to the physical and conformational heterogeneity of protein produced by recombinant DNA. We have made extensive modifications of the Rev protein in order to improve its solubility for structural work. In addition, we are attempting to further stabilize Rev by forming binary complexes with either antibodies or tubulin. To generate a wider range of monoclonal antibodies for co-crystallization studies, we have made recombinant antibodies based on phage display selection. Bone marrow from immune rabbits was used to create an antibody library. Rev binding monoclonal antibodies (mAbs) were selected then humanized producing chimeric mAb fragment antigen binding portions (Fab). These Fabs contain rabbit variable domains and human constant domains and were produced in bacteria using an E.coli secretion system. One such produced Fab bound to HIV-Rev with high affinity and depolymerized the normally highly associated Rev protein forming physically homogenous low molecular weight immune complexes. These complexes appear suitable for crystallization screening. High affinity humanized anti-Rev Fab may also have potential therapeutic use. (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 high affinity binding between these proteins. Using protein engineered variants of Nef and Tat we have prepared complexes for more detailed structural studies (3) HIV protease, a homodimeric protein is essential in the viral life cycle and a major anti-HIV drug target. Peptides derived from the N- and C-terminal regions of the HIV-1 protease dimer interface inhibit protease activity by preventing dimerization (monomeric protein is inactive). In previous work, it was shown that the solubility and cell permeability of the peptides was enhanced by linking the transduction domain of HIV-Tat. Methods for studying the mechanism of the dimerization inhibition were developed and this work was facilitated by the use of multidrug resistant HIV-proteases with additional mutations to facilitate biophysical analysis (produced by expression in recombinant bacterial cells).
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