Characterization of peptide substrates and viral enzyme that affect the cleavage site specificity of the human spumaretrovirus proteinase

Oligopeptides that correspond to proteolytic cleavage site junctions of the native Gag and Pol proteins are specifically cleaved by retroviral asparta te proteases (PRs). The role of the flap subdomain of the PR of the human s pumaretrovirus (HSRV) and of substrate peptides in cleavage site specificit y was analyzed by site-directed mutagenesis. Native and mutant peptides wer e subjected to proteolysis by the authentic and mutated recombinant viral e nzyme. The results reveal that Glu residue 54 of the HSRV PR is an essentia l specificity determinant for proteolytic processing of the structural prot eins. Peptides that represent in vivo cleavage sites were susceptible to pr oteolysis by the recombinant HSRV PR, but one peptide located at the juncti on between the PR and reverse transcriptase domains was completely resistan t to cleavage. Thus the data indicate that a proteolytic cleavage between t hese domains does not occur in vivo. Naturally occurring and mutant forms o f the cleavage-resistant peptide were therefore analyzed by circular dichro ism to determine if differences existed in the secondary structures of the peptides that did or did not serve as substrates. The data show that differ ences in the secondary structure of the native and mutant peptides analyzed does not seem to play a crucial role for cleavage site specificity in HSRV PR. Instead highly conserved hydrophobic residues at distinct positions of the HSRV cleavage site junctions contribute to the specificity observed as reported for HIV-1 PR.