Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors.
Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.
Leung
D
D
Centre for Drug Design and Development, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.
Schroder
K
K
White
H
H
Fang
N X
NX
Stoermer
M J
MJ
Abbenante
G
G
Martin
J L
JL
Young
P R
PR
Fairlie
D P
DP
D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; C080102:NS2B protein, flavivirus; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins