Fig. 5.

Effects of E protein stem mutations on the assembly and infectivity of replicon particles. (A) DENV2 replicon particles were prepared by sequential transfections of BHK-21 cells with replicon RNA and WT or mutant CprME expression vector. At 30 h after the first transfection, the cells were examined for E protein expression by Western blot analysis using anti-E MAb 4G2. Host γ-tubulin protein was used as a loading control. (B) At 32, 48, and 72 h posttransfection, the luciferase activities in the transfected cells were measured. (C) Viral RNA extracted from the culture supernatants (containing replicon particles) was quantified by real-time RT-PCR; the relative amounts of replicon RNA to the amount of the WT replicon RNA at 72 h posttransfection (100%) are presented. (D) Equal amounts of WT and mutant replicon particles (normalized by RNA copy numbers) were inoculated into Vero cells. At 48 h, the luciferase activities in the infected cells were measured; the luciferase signals relative to those of WT replicon particle-infected cells (100%) are shown. Data are means and standard errors from duplicate experiments.