Intrinsic activity of precursor forms of HIV-1 proteinase.
The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.
Phylip
L H
LH
Department of Biochemistry, University of Wales College of Cardiff, UK.
Mills
J S
JS
Parten
B F
BF
Dunn
B M
BM
Kay
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data
D004277:DNA, Single-Stranded; D004792:Enzyme Precursors; D016333:HIV Protease