ANALYSIS OF SUBSTRATE CLEAVAGE BY RECOMBINANT PROTEASE OF HUMAN T-CELL LEUKEMIA-VIRUS TYPE-1 REVEALS PREFERENCES AND SPECIFICITY OF BINDING

Human T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expre ssed in bacteria and purified by gel filtration. A continuous spectrop hotometric assay was used to measure the kinetic parameters of substra te hydrolysis by PR14. Several peptide substrates containing HTLV-1 se quences known to be cleaved by PR14 were used. Cleavage analysis showe d that the affinity with which PR14 binds these substrates is higher t han that previously reported for HTLV-1 Gag peptides. Also, the affini ties of peptides containing the sites involved in autocleavage of prot ease from its precursor are higher than for the peptides containing si tes required for structural protein maturation. This suggests that the autocatalysis of protease from its own precursor has priority over ot her cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases. As the N- and C-terminal regions of retroviral aspartic proteases are known to cont ribute to stability of the dimer by forming antiparallel beta-strands, short peptides corresponding to these terminal sequences of HTLV-1 pr otease were tested for their ability to inhibit cleavage of substrates by PR14. Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, wherea s a peptide containing a sequence situated further from the C terminus was less effective. An inhibitor of the protease of human immunodefic iency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR 14.