title: Characterization of the West Nile virus protease substrate specificity and inhibitors.
abstract: West Nile virus (WNV), a mosquito-borne member of Flaviviridae, is a human pathogen causing widespread disease for which there is no vaccine or chemotherapy. The two-component viral serine protease consists of a heterodimeric complex between the hydrophilic domain of the cofactor, NS2B (NS2BH) and the protease domain (NS3-pro). The protease is essential for polyprotein processing followed by assembly of viral replicase and genome replication. Therefore, the protease is an excellent target for development of antiviral therapeutics. Here, we report the expression in Escherichia coli, purification, and characterization of biochemical and kinetic properties of the WNV protease. Furthermore, we show that the WNV and the dengue virus type 2 (DENV-2) proteases are inhibited by aprotinin with inhibitor constants of 0.16 and 0.026 microM, respectively. Molecular modeling of the WNV protease/aprotinin complex, based on the known crystal structures of the WNV NS2BH-N3pro and aprotinin, suggest a potentially strong interaction between the P2 Lys and the protease activator peptide, NS2BH. This conclusion based on molecular modeling is in agreement with our data of a higher k(cat)/Km value with the substrate, Boc-Gly-Lys-Arg-MCA than the Boc-Gly-Arg-Arg-MCA and is also consistent with the results of an earlier study that were based on substrate-based inhibitor peptides.
authors.affiliation: Department of Microbiology and Immunology, Georgetown University School of Medicine, 3900 Reservoir Road NW, Med-Dent SW309, Washington, DC 20057, USA. nhm3@georgetown.edu
authors.forename: Niklaus H
authors.initials: NH
authors.lastname: Mueller
authors.forename: Changsuek
authors.initials: C
authors.lastname: Yon
authors.forename: Vannakambadi K
authors.initials: VK
authors.lastname: Ganesh
authors.forename: R
authors.initials: R
authors.lastname: Padmanabhan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D003716:Dengue Virus / Q000187:drug effects / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D046912:Multiprotein Complexes; D009842:Oligopeptides / Q000737:chemistry; D020365:RNA Helicases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014902:West Nile virus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D004279:DNA, Viral; D046912:Multiprotein Complexes; D009842:Oligopeptides; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases