Cleavage activity of hepatitis C virus serine proteinase.
To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.
Kakiuchi
N
N
HQL Research Labs., Sumitomo Metal Industries Ltd., Kyoto. nkakiuch@virus.kyoto-u.ac.jp
Komoda
Y
Y
Hijikata
M
M
Shimotohno
K
K
D002413:Cations, Divalent; D002846:Chromatography, Affinity; D016174:Hepacivirus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000494:pharmacology
D002413:Cations, Divalent; C093542:NS4 protein, hepatitis C virus; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; D012697:Serine Endopeptidases