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. The solution structure of Nef was completed last year. Based on the structure, we have made several deletion and site-directed mutations that improve the physical properties of the protein. We are now investigating some of the specific protein-protein interactions involving Nef. One important interaction is with the cytoplasmic tail of CD4. The N-terminal domain of the lck Scr tyrosine kinase also binds to this region. Nef may, thus, exert its biological effect by displacement of the lck Scr tyrosine kinase. To gain further insights into these interactions, we have expressed the N-terminal portion of the lck and the cytoplasmic tail of CD4 and following further characterization of these protein structural studies will be initiated. (2) HIV Rev is an important regulatory factor required for HIV expression. This RNA binding protein has a strong tendency to self-associate into very high molecular weight fibrous polymers. Using these fibers, spectroscopic methods and cryo-electron microscopy and image analysis were used to obtain a low- resolution model of the protein. To extend this structural information using higher resolution methods, such as X- crystallography and NMR, solution conditions have been explored to maintain the protein in a physically homogeneous, non- aggregated, low molecular weight state. Non-denaturing and relatively mild solvents systems were found which prevented protein polymerization and aggregation. However, under all conditions tested, the non-polymerized protein appears to be only partially folded state as determined by NMR. To stabilize a non-polymerizing Rev, we are have constructed a series of fusion proteins that include both full-length and C-terminally deleted Rev with various fusion partners. These fusion proteins are currently under investigation (Steven, Watts, Bax). (3) In our continuing studies on the HIV protease, novel mutants designed to stabilize it against proteolytic degradation have been made. These mutants will facilitate structural-function studies by NMR based methods (London). Other protease mutants have been made to study the role of oxidative inactivation (Davis). (4) MAP30 is an anti-HIV plant protein that appears to inhibit HIV-1 integrase and other essential viral enzymes. Purification methods and biosynthetic labeling protocols for this 30 kDa protein were developed a few years ago and we have continued to support its structural determination by high-resolution NMR methods. The structure has been completed over the last year and is currently being refined prior to publication (Torchia, Wang and S.Lee-Huang). (5) The assembly of HIV is mediated by the gag gene product (p55). Shell. The gag polyprotein is cleaved by HIV-1 protease into four subunits: p17 (matrix protein), p24 (capsid protein), p7 (NC) and C-terminal p6. The cleaved product forms mature viral capsids. The mature viruses have a cone- or rod-shaped appearance. The organization and overall morphology of the HIV nucleocapsid has not yet been clearly established. We intend to produce p55 and study the maturation of this protein in-vitro, monitoring the assembled capsid structures by biophysical and high-resolution electron microscopy. We have initiated this work by making several of the individual components: p24, p17 and p7. In a related study we are investigating specific protein-protein interactions involving the N-terminal domain of p17 and the cytoplasmic tail of gp41. For this work we have expressed the ~150 residue cytoplasmic regions of both SIV and HIV gp41. Although the p17 can be expressed and purified with relative ease, the gp41 moieties have proved very problematic. We have obtained these proteins as GST-fusion proteins and are currently examining their chemical and physical properties. 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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