PROTEOLYTIC PROCESSING OF PARTICLE-ASSOCIATED RETROVIRAL POLYPROTEINSBY HOMOLOGOUS AND HETEROLOGOUS VIRAL PROTEINASES

Retroviral proteinase(PR)-catalyzed cleavage of the viral Gag and Gag- Pol polyproteins within the nascent virus particle is required for pro ductive viral infection. Kinetic characterization and specificity anal yses have been reported for several retroviral PR using oligopeptide s ubstrates. In this study, we performed a comparative analysis of PR fr om avian, bovine, simian and human retroviruses using polyproteins of human immunodeficiency virus (HIV) type 1 or avian leukosis virus as s ubstrates. Polyproteins were derived from immature virus-like particle s purified from culture medium of transfected or recombinant baculovir us-infected cells. Specific cleavage to the correct size intermediate and end products occurred in the presence of detergent and homologous PR. HIV-1 PR cleaved its Gag precursor to completion at a concentratio n of approximately 25 nM but cleaved the Gag-Pol precursor incompletel y even at fourfold higher PR concentration. In contrast to the require ment for high ionic strength for peptide cleavage reported previously, we found that Gag protein cleavage by HIV-1 PR proceeded best at low ionic strength, for both of the protein substrates tested. HIV-2 PR wa s approximately sixfold less active than HIV-1 PR. PR from avian myelo blastosis-associated virus (MAV) yielded efficient cleavage of the HIV -1-polyprotein only at concentrations above 1 mu M. Both enzymes were stimulated by high salt and their cleavage products were identical or very similar to those of HIV-1 PR. A mutant of MAV PR engineered to cl eave HIV-1 peptide substrates did not cleave the HIV-1 polyprotein at a concentration of 0.4 mu M. The PR of Mason Pfizer monkey virus cleav ed this polyprotein very poorly, whereas PR of bovine leukemia virus c leaved it, albeit at different sites.