Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease.
West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I <0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 approximately 1 microm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.
Nall
Tessa A
TA
Centre for Drug Design and Development, Institute for Molecular Bioscience, Univeristy of Queensland, Brisbane, Australia.
Chappell
Keith J
KJ
Stoermer
Martin J
MJ
Fang
Ning-Xia
NX
Tyndall
Joel D A
JD
Young
Paul R
PR
Fairlie
David P
DP
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D003593:Cytoplasm / Q000378:metabolism; D003716:Dengue Virus / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D012965:Sodium Chloride / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014902:West Nile virus / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D005990:Glycerol; D008024:Ligands; D010455:Peptides; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D012965:Sodium Chloride; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases