We have investigated the mechanism of autoprocessing of the human immunodeficiency virus, type 1, protease from a polyprotein that contains flanking sequences at the protease's termini [MBP-12AA-PR-19AA]. The dimeric MBP-12AA-PR-19AA protein has an active site similar to that of the mature enzyme and can bind inhibitors and substrates, although its enzymatic activity is much lower than that of the mature protease. The N-terminal cleavage is an intramolecular reaction that occurs much faster than the C-terminal cleavage and leads to the accumulation of the intermediate protein, PR-19AA (13.2 kDa). The 13.2 kDa protein was purified to near homogeneity by chromatography on a size exclusion column under denaturing conditions. A comparison of inhibition constants, kinetic parameters for the hydrolysis of a peptide substrate, and pH rate profiles indicate that the 13.2 kDa protein has an active site that is similar to that of the mature protease. The major difference between the 13.2 kDa protein and the mature enzyme is in their susceptibility to urea denaturation. Initial rates of the conversion of the 13.2 kDa protein to the mature protease is dependent on the square of the protein concentrations indicating that the C-terminal sequence is cleaved via an intermolecular mechanism in the autoprocessing reaction. To compare the interface contacts of the homodimeric protease fusion protein with that of the mature protease we have expressed and purified an inactive fusion protein. This fusion protein inhibits the mature wild-type protease through heterodimer formation. In retroviral assembly, RNA selection and packaging involves the two cysteine arrays of the nucleocapsid (NC) protein which form zinc fingers. We demonstrate specific cleavage within the first finger of the HIV-1 NC protein by the viral protease that is dependent upon removal of zinc ions. The reaction is time dependent and is completely inhibited by 1 mM pepstatin A, a specific inhibitor of aspartic acid proteases. Also, 3-nitrosobenzamide, a compound shown to have antiviral properties, and cupric ions, that release zinc through oxidation of the cysteine residues of the finger, render the NC protein resistant to cleavage.
