A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis
Fig 2
Effect of virion maturation state on neutralization sensitivity of WNV E T198F RVPs.
(A) We assessed the level of V5-tagged prM in WNV RVPs prepared using standard (Std) conditions, or those increasing the efficiency of prM cleavage (Furin) by SDS-PAGE and Western blotting of pelleted virions using a mouse monoclonal antibody against V5 (top panel). The level of E protein (bottom panel), as detected by mouse monoclonal antibody, 4G2, was used as a loading control for each RVP preparation. Data are representative of three independent experiments performed with independent RVP preparations. (B) We concurrently tested Std and Furin preparations of WT and T198F RVPs for sensitivity to neutralization by mAb E60. Representative dose-response neutralization curves are shown, with the y- and x-axes representing percent infectivity and mAb concentration, respectively. Infectivity was normalized to levels observed in the absence of antibody. Error bars indicate the range of infectivity from duplicate infections. (C) Mean EC50 values for E60 against Std or Furin preparations of WT and T198F RVPs. Error bars represent the SEM. The indicated p-values were obtained from paired t-tests. (D) Mean percentages of Std or Furin WT and T198F RVPs resistant to neutralization at the highest concentration of E60 tested (10 μg/ml). Error bars represent the SEM. The indicated p-values were obtained from paired t-tests. For (C) and (D), mean values were obtained from three independent experiments performed in duplicate using independent RVP preparations.