Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain.

The mature human immunodeficiency virus type 1 proteinase (PR; 11 kDa) can cleave all interdomain junctions in the Gag and Gag-Pol polyprotein precursors. To determine the activity of the enzyme in its precursor form, we blocked release of mature PR from a truncated Gag-Pol polyprotein by introducing mutations into the N-terminal Phe-Pro cleavage site of the PR domain. The mutant precursor autoprocessed efficiently upon expression in Escherichia coli. No detectable mature PR was released; however, several PR-related products ranging in size from approximately 14 to 18 kDa accumulated. Products of the same size were generated when mutant precursors were digested with wild-type PR. Thus, PR can utilize cleavage sites in the region upstream of the PR domain, resulting in the formation of extended PR species. On the basis of active-site titration, the PR species generated from mutated precursor exhibited wild-type activity on peptide substrates. However, the proteolytic activity of these extended enzymes on polyprotein substrates provided exogenously was low when equimolar amounts of extended and wild-type PR proteins were compared. Mammalian cells expressing the mutated precursor produced predominantly precursor and considerably reduced amounts of mature products. Released particles consisted mostly of uncleaved or partially cleaved polyproteins. Our results suggest that precursor forms of PR can autoprocess but are less efficient in processing of the Gag precursor for formation of mature virus particles.