Cleavage of RT/RNase H by HIV-1 Protease and Analysis of Substrate Cleavage Sites in vitro

Abstract

The pol-gene of HIV-1 expressed in bacteria undergoes autocatalytic processing which results in the generation of the p66 reverse transcriptase (RT)/RN ase H, the p32 endonuclease and the p9 protease. The partially purified protease generates in vitro from the p66 molecule a p66/p51 heterodimer, typical of the normally observed RT/RNase H, and a p15 carboxyterminal fragment. A synthetic peptide AETF’YVD derived from the p51/p15 junction is cleaved by the protease in vitro and may represent the natural cleavage site. The RT/RNase H activities associated with p66 and p51 and p66/p51 heterodimers were determined after renaturation of these proteins from Polyacrylamide gels. The p66/p51 heterodimers exhibit about eightfold higher RT and RNase H activities than each of the individual subunits alone suggesting that heterodimers are of biological relevance. Protease activity was monitored by three assays, by cleavage of denatured ovalbumin, of the MS2-gag fusion protein or synthetic peptides. Several synthetic peptides representing various poten tial protease cleavage sites and modifications thereof were analyzed in vitro for their efficiency of cleavage by the protease. One synthetic peptide representing the natural amino-terminal cleavage site of the protease was modified by several amino acid substitutions in order to characterize the specificity of the protease. Several of the peptides as well as cerulenin and acetyl-pepstatin were analyzed as protease inhibitors.