Purified WNV DIII. The construction, expression, purification and refolding of DIII of WNV E protein is described in greater detail elsewhere (G.N., T.O., M.D. & D.F., unpublished data). Briefly, wild-type, N394K and K307N DIII were generated from an infectious cDNA clone of the New York 1999 strain of WNV (gift of R. Kinney, Centers for Disease Control and Prevention, Fort Collins, CO) using PCR and Quik-change mutagenesis (Stratagene). After cloning into a PET21 vector (Novagen) and sequence confirmation of the mutations, we transformed plasmids into BL21 Codon Plus E. coli cells (Stratagene). Bacteria were grown in Luria broth, induced with 0.5 mM isopropyl thiogalactoside (IPTG) and pelleted. Subsequently, we lysed bacteria after the addition of lysozyme, sonicated them and recovered DIII as insoluble aggregate from the inclusion bodies. DIII was denatured in the presence of guanidine hydrochloride and β-mercaptoethanol and refolded by slowly diluting out the denaturing reagents in the presence of L-arginine, EDTA, PMSF, reduced glutathione and oxidized glutathione. We separated refolded DIII from aggregates on a Superdex 75 16/60 size-exclusion column (Amersham Bioscience) and concentrated it using a centricon-10 spin column into 20 mM Hepes pH 7.4, 150 mM NaCl and 0.01% NaN 3 . After refolding, wild-type DIII reacted with all domain III-specific monoclonal antibodies including those that recognized conformationally sensitive epitopes.