Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease.

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.