Fig 3.

Rapid mutagenesis of the prM gene of WNV using pWNV-GFP-backbone. A panel of viruses encoding single-amino-acid substitutions in the 92 amino acids of the “pr” portion of the prM protein was constructed. PCR fragments encoding an alanine mutation at each position were generated by OE-PCR; alanine residues of prM were replaced by glycine substitutions. Each PCR fragment was ligated into pWNV-GFP-backbone V3 and transfected into HEK-293T cells. Virus-containing supernatants were harvested at 72 h posttransfection; the titer was determined as described in the legend of Fig. 2. (A) Production of the WT and representative prM variants N15A, G42A, and N60A. Data from representative prM variants are shown and color coded to correspond to the data presented in the bottom panel. (B) The infectious titer of each prM variant was determined from linear portions of the virus dose-infectivity curve. Infectivity is expressed as the titer of each variant relative to those determined in paired experiments performed with the WT virus (×100%). In cases where independent preparations of virus were produced, error bars represent standard errors.