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 (indicated below in parentheses) 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) Nef is a 23 kDa protein essential for the pathogenic properties of HIV. We are investigating some of the specific protein-protein interactions involving Nef especially interaction with the 39-residue cytoplasmic tail of CD4. We have devised novel expression systems for producing the small CD4 domain that are useful in general for producing small and relatively unstable peptides. To study the interaction using NMR, the Nef protein has been modified to enhance its solubility. Apart from studying bimolecular complexes we are also preparing fusion proteins consisting of Nef and CD4 tethered by flexible loop regions. Our attempts to produce and formulate these proteins for NMR studies continue. (2) HIV Rev is an important regulatory factor required for HIV expression. A novel interaction between HIV-1 Rev and microtubles (MTs) has been discovered. This results in the formation of bilayered rings that are 44-49 nm in diameter and 3.4 - 4.2 megadalton in mass. The interaction involves the amino terminal-domain of Rev and the face of tubulin exposed on the outer surface of the MTs. The overall results suggest that Rev may interact in-vivo with MTs, thus, inducing destabilization. This is consistent with previous findings that describe the disruption of MTs following HIV-1 infection. (3) MAP30 is a plant protein with anti-HIV and anti-tumor activities. Purification methods and biosynthetic labeling protocols for this 30 kDa protein were developed for NMR structure determination. (Torchia, Bax, Wang and S.Lee-Huang). The high-resolution structure (published: Cell November 1999) revealed that MAP30 adapts the ricin A chain fold with secondary structure elements similar to those observed in ricin A chain and other ribosome inactivating proteins. Subsequent biochemical assays (Pommier) showed that MAP30 acts as a DNA glycosidase/ ap lyase. This activity could account for MAP30 inhibition of HIV-1 integrase as well as explaining its anti HIV/anti-tumor activities. We are currently making site-directed mutants of those residues suggested to be critical in the proposed depurination and ap lyase activities. The proteins are currently being biochemically characterized. 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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