Mutagenesis of protease cleavage sites of the HIV-1 gag polyprotein.
Int Conf AIDS 1990 Jun 20-23; 6:324 (abstract no. 1040) Tritch R, Cheng YS, Viitanen P, Yin F, Erickson-Viitanen S; Medical Products Department, E.I. Du Pont de Nemours and Co., Inc., Wilmington, Delaware, USA
The virally encoded protease of HIV is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, and integrase, and the core proteins from the gag-pol and gag polyprotein precursors, respectively. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield first p15, followed by p6 and p9, followed by p17 and finally mature p24. We have placed unique restriction sites flanking the p17-p24 and p24-p15 domains in order to facilitate replacement of cleavage site sequences utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved Met-Met at the p24-p15 junction with Tyr-Pro, or replacement of the Tyr-Pro at the p17-p24 junction with Met-Met results in sites that cannot be efficiently cleaved. Addition of basic residues near the p17-p24 junction or replacement of this junction with symmetrical amino acid sequences result in intermediate cleavage rates. Our results suggest that although the majority of specificity determinants reside within the four amino acids immediately flanking the cleavage pair, more distant residues also affect the cleavage rate.
Keywords: AEGIS, Gene Products, gag, HIV Protease, HIV-1, Endopeptidases, RNA-Directed DNA Polymerase, Mutagenesis, Site-Directed, In Vitro, ICA6
