Expression of WNV E protein on yeast. The ectodomain or DIII of WNV E protein was expressed on the surface of yeast using a modification of a previously described protocol for surface expression of T cell receptors 29 . Amino acid residues 1-415 (ectodomain) or 296-415 (DIII) of WNV E protein were amplified with BamHI and XhoI sites at their 5´ and 3´ ends, respectively, by PCR from the New York 1999 infectious cDNA clone (R. Kinney, Centers for Disease Control and Prevention, Fort Collins, CO). The resulting products were digested with BamHI and XhoI, and cloned as downstream fusions to the yeast Aga2 and Xpress epitope tag genes in the yeast surface display vector pYD1 (Invitrogen). An upstream GAL1 promoter controls fusion protein expression. These constructs were transfected into the S. cerevesiae yeast strain EBY100 (refs. 25,41) resulting in yeast that expressed the WNV E ectodomain or DIII. Yeast that only expressed the Xpress epitope tag linked to Aga2 were prepared in parallel by transfecting EBY100 cells with the parent vector pYD1. Individual yeast colonies were grown to log phase overnight in tryptophan-free media containing 2% glucose at 30 °C and harvested in log phase. Fusion protein expression was induced on the yeast surface by growing yeast for an additional 24 h in tryptophan-free media containing 2% galactose at 25 °C. We harvested yeast, washed them with PBS supplemented with 1 mg/ml BSA and immunostained them with 50 µl of monoclonal antibody (25 µg/ml) against the Xpress tag or WNV E protein. After 30 min, we washed yeast three times and stained them with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (Molecular Probes). Subsequently, the yeast cells were analyzed on a Becton Dickinson FACSCaliber flow cytometer.