Production Of Hiv And Hiv Related Proteins For Structura

Arthritis, Musculoskeletal, Skin Dis

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Abstract

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. This work is still in progress and we are continuing to modify both Nef and CD4 to facilitate the study. (2) 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. (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.