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 recently. 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 in E.coli. Our attempts to produce and formulate these proteins for NMR studies continue. (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. We have continued our efforts to gain more structural information by focusing on smaller regions of the protein, especially the N-terminal half that mediates binding to its target RNA (RRE). Fusion proteins have been expressed in bacteria and their characterization continues.(3) In our continuing studies on the dimeric HIV protease, we have studied the reversible oxidation of residue 95 and suggest this regulates the activity of both HIV type I and type II enzyme. Residue 95 is cysteine (type I) or methionine (type II) and oxidation of these sulphur containing amino acids, located at the dimer interface, inhibits activity in an reversible manner. These studies may be useful as a model of redox regulation of the HIV-protease whose function may be regulated in cells under oxidative stress (Davis). Work has also been carried out on the proteolytic processing of the HIV-1 protease precursor. For these studies the HIV-1 protease linked to the native transframe region was expressed in E.coli (TFR-PR). Using the TFR-PR construct, the mechanism of the autocatalytic maturation was studied. Cleavage at the N-terminus of the protease leads to stabilization of the dimeric structure (Louis). (4) 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 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. (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 have produced p55 and intend to 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 expressing in bacteria 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 continue to examine their chemical and physical properties in an attempt to formulate them for structural work. 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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