A comparative biochemical analysis of the NS2B(H)-NS3pro protease complex from four dengue virus serotypes.
The two-component protease NS2B-NS3 of dengue virus mediates proteolytic processing of the polyprotein precursor and therefore represents a target for the development of antiviral drugs. The amino acid sequences of the NS3 serine protease and the NS2B cofactor exhibit relatively low degrees of conservation among the 4 serotypes thus suggesting that differences in enzyme activity exist which could modulate their susceptibility to future protease inhibitors. In this study we have addressed the question of functional similarity among the NS2B(H)-NS3pro proteases from 4 dengue virus serotypes by employing a uniform approach to clone, purify and assay proteolytic activity of these enzymes. Significant differences were observed for patterns of protein formation and expression levels in the E. coli host. Renaturation of the NS2B(H)-NS3pro precursors from dengue virus serotypes 2, 3 and 4 mediated by artificial chaperone-assisted refolding yielded enzymatically active proteases, whereas the enzyme from serotype 1 was obtained as soluble protein. Kinetic experiments using the GRR-amc substrate revealed comparable K(m) values while k(cat) values as obtained by active-site titration experiments displayed minor variations. Denaturation experiments demonstrated significant differences in half-life of the NS3 proteases from serotypes 2, 3 and 4 at 50 degrees C, whereas pH optima for all 4 enzymes were comparable.
Iempridee
Tawin
T
Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phutthamonthon 4 Road, Nakornpathom 73170, Thailand.
Thongphung
Ratchanu
R
Angsuthanasombat
Chanan
C
Katzenmeier
Gerd
G
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003716:Dengue Virus / Q000145:classification / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012703:Serotyping / Q000145:classification; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000085242:Viral Proteases
D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases