Over the past 2-1/2 years we have (i) determined that low-molecular weight, virus-derived HIV-1 gag proteins p1, p2 and p6 lack regular tertiary structure in solution, (ii) demonstrated that the conserved """"""""CCHC arrays"""""""" (CCHC = Cys-X2-Cys-X4-His-X4-Cys; X = variable amino acid) of p7 (nucleocapsid protein, NC) are populated with zinc in intact virions, providing direct evidence for the physiological relevance of CCHC zinc fingers, (ii) determined the three-dimensional solution-state structure of the intact, virus-derived HIV-1 NC protein, (iii) identified a sequence-dependent single-stranded nucleic acid binding mode for the N-terminal CCHC zinc finger and determined the structure of a zinc finger-nucleic acid complex, and (iv) identified a new class of anti-HIV agents that function by ejecting zinc from HIV-1 NC zinc fingers. Having nearly completed studies of the low-molecular weight gag proteins, we intend to focus future efforts on the structure determination of the HIV-1 capsid protein (CA). HIV-1 CA will be expressed and purified using a novel procedure developed by Dr. Debouck and co-workers at SmithKline Beecham that involves simultaneous expression of the HIV protease and a derivative of the gag precursor protein p55 in E. coli. The protein is processed by the protease in vivo and subsequently isolated and purified under non-denaturing conditions. Biophysical data and 1/H NMR studies indicate that the recombinant protein is folded in solution and amenable to high- resolution NMR-based structural studies. 15/N- and 13/C, 15/N- Isotopically labeled samples will be prepared for multi-dimensional double- and triple-resonance NMR experiments, and interproton distance estimates derived from nuclear Overhauser effect data will be employed for 3D solution-state structure calculations. Knowledge of the structure and biophysical properties of the HIV capsid protein is important for understanding molecular-level aspects of virus assembly and infectivity and should facilitate the development of potential chemotherapeutic agents designed to interfere with capsid formation and virus assembly.
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