Methods of enzymology, chemical and enzymatic kinetics, and synthetic, physical and analytical chemistry are being used to investigate the mechanisms of action of reverse transcriptase (RT) and protease enzymes of HIV-1, with the ultimate goal of developing specific inhibitors for these enzymes. Optical assays for retroviral proteases and P's, developed in this laboratory, are being employed in studies of the effects of reaction conditions and potential inhibitors on the activity of both RT and retroviral proteases. i) A continuous UV spectrophotometric assay described in a previous Report was used to study the elongation, catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I, of a partially self-complementary """"""""hairpin"""""""" shaped oligonucleotide consisting of 35 mononucleotide residues. The sensitivity of the method permits the measurement of incorporation of a single base. ii) Concentrations of sodium chloride up to 4.5 M accelerated the hydrolysis of a chromogenic peptide substrate catalyzed by the protease of avian myeloblastosis virus. Similar effects have been observed by us for the cleavage of a peptide substrate catalyzed by HIV-1 protease, and have also been reported by others for cleavage of several peptides by this enzyme. These salt effects appear to be unrelated to the association of subunits that is required for the activity of retroviral aspartyl proteases, since very similar behavior is observed with the monomeric, mammalian enzyme pepsin. iii) We have identified two amino-acid repeat motifs in RT which are conserved in ten HIV-1 and eight HIV-2 isolates, and which occur in a region of the protein that may be involved in association of the p66 and p51 subunits of this enzyme. One motif is a modified """"""""leucine zipper"""""""", whereas the other motif involves five tryptophans. Since the activity of HIV-1 RT has been suggested to require subunit association, an understanding of this process may lead to novel approaches to the inhibition of this enzyme.
