Abstract
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.
Highlights
The West Nile Virus (WNV)1 was first detected in a woman living in the West Nile region of Uganda almost 70 years ago [1]
By analogy with the homologous Dengue virus NS3 protease, for which our truncation studies had previously shown that a 40-residue hydrophilic domain of NS2B is necessary for catalytic activity of the NS3 protease, [6] we infer below that a similar sequence within the WNV NS2B is likely necessary for catalytic activity of the WNV NS3 protease
Precedents for inhibitors of viral proteases being potential antiviral drugs lie in the success of inhibitors of proteases of the human immunodeficiency viruses (HIV), currently the most effective treatments for humans with HIV/AIDS [10, 11], and in the early promise being shown by inhibitors of the NS3 protease of hepatitis C virus [12]
Summary
The West Nile Virus (WNV)1 was first detected in a woman living in the West Nile region of Uganda almost 70 years ago [1]. This article describes the design and construction of a catalytically active recombinant WNV NS2B-NS3 protease enzyme construct, the characterization of factors that influence enzymatic processing of short synthetic chromogenic substrates containing para-nitroanilide (pNA), the first reported inhibitor of WNV protease, and the development of a three-dimensional homology-modeled structure of the WNV NS3 protease based on the known crystal structures of Dengue NS3 protease and HCV NS3 protease complexed with cofactor NS4A.
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