FIG 6.
A single point mutation in the flavivirus E ij loop inhibits ZIKV infectious particle production and impacts envelope protein accumulation. (A) ZIKV MR766 envelope (PDB ID 5JHM) and CHIKV E1 (PDB ID 3N42). The ij loop and ZIKV A250 and CHIKV A226 residues are in red. (B) 293T cells were transfected with WT ZIKV or ZIKV EA250V infectious clone plasmids, and virus supernatants were collected at 48 hpi. Virus titers were quantified by plaque assay. n = 2 with internal technical duplicates. Extracellular (C) and intracellular (D) ZIKV RNA was quantified by RT-qPCR at 72 h posttransfection. RNA is normalized to WT ZIKV. There were three independent experiments with internal technical duplicates. (E) 293T cells were transfected with WT ZIKV or ZIKV EA250V plasmids. Cells were harvested at 72 h posttransfection and lysed in Laemmli buffer, and ZIKV proteins were analyzed by SDS-PAGE and immunoblotting. The immunoblot is representative of at least 3 independent transfections.