Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding

Abstract

Human T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expressed in bacteria and purified by gel filtration. A continuous spectrophotometric assay was used to measure the kinetic parameters of substrate hydrolysis by PR14. Several peptide substrates containing HTLV-1 sequences known to be cleaved by PR14 were used. Cleavage analysis showed that the affinity with which PR14 binds these substrates is higher than that previously reported for HTLV-1 Gag peptides. Also, the affinities of peptides containing the sites involved in autocleavage of protease from its precursor are higher than for the peptides containing sites required for structural protein maturation. This suggests that the autocatalysis of protease from its own precursor has priority over other cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases. As the N-and Cterminal regions of retroviral aspartic proteases are known to contribute to stability of the dimer by forming antiparallel fl-strands, short peptides corresponding to these terminal sequences of HTLV-1 protease were tested for their ability to inhibit cleavage of substrates by PRI4. Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, whereas a peptide containing a sequence situated further from the C terminus was less effective. An inhibitor of the protease of human immunodeficiency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR14.

Stabilization from Autoproteolysis and Kinetic Characterization of the Human T-cell Leukemia Virus Type 1 Proteinase

“…The difference in K m values may be because of the introduction of the bulky aromatic residue in the P1Ј position. This may also be the reason why the naturally occurring cleavage site sequences representing the autocatalytic maturation sites (TF1/PR and PR/p3) in HTLV-1 as compared with the corresponding chromogenic derivatives do not have substantially higher affinities toward HTLV-1 PR than the peptides representing other sites, as noted for the chromogenic derivatives by Daenke et al (16). The reason for the differences in the reported k cat values is less obvious.…”

“…The specific activity reported using the chromogenic substrate representing the CA/NC site (1 mol/min/mg; Ref. 16) suggests a much smaller catalytic rate.…”

“…This can be attributed to the difficulty in protein purification and to the autoproteolysis that is known to occur with native retroviral proteinases (19,20). Peptides representing some of the natural cleavage sites of HIV-1 are hydrolyzed by HTLV-1 PR (16,18), although no kinetic parameters were reported for these peptides.…”

Identification of peptidomimetic HTLV-I protease inhibitors containing hydroxymethylcarbonyl (HMC) isostere as the transition-state mimic

“…Both the recombinant and synthetic HTLV-I PR analogs specifically hydrolyzed synthetic peptide substrates, APQVL*PVMHP (p19/24), KTKVL*VVQPK (p24/15) and APQVL*NphVMHPL (the modified p19/24) (* indicate scissile bond), 26,27 and pepstatin A, which is known as a typical aspartyl PR inhibitor, showed a moderate inhibitory activity with K i values of 14.6 and 36.5 lM against rec-and syn-HTLV-I PRs, respectively (Tables 1 and 2). 28,29 These K i values were almost equal to that of previously reported recombinant HTLV-I PR (K i = 17lM).…”

Expression, Characterisation and Mutagenesis of the Aspartic Proteinase from Equine Infectious Anaemia Virus

“…The compound Ro31-8959 (16 in Table 4) is a potent inhibitor of proteinase from HIV-1 and HIV-2 [24] and from SIV [25] but was found to be ineffective against wild-type EIAV proteinase. Similarly, it did not inhibit purified proteinases from HTLV-1 [26] and FIV (B. M. D. and D. J. P., unpublished observation) and was unable to impair the replication cycle of a number of other retroviruses [27]. Proteinases from the three susceptible viruses, HIV-1, HIV-2 and SIV, all have a Gly at position 48 ( Fig.…”