Figure 3. cHP mutants do not progress to late rounds of translation.

BHK cells were transfected with a DENV2 reporter replicon and translation was assessed at the given timepoints by luciferase assay. (A) Schematic diagram of pDRrep, a reporter replicon of DENV2 strain 16681, in which most of the structural protein-coding region has been replaced by Renilla luciferase (Rluc). The N-terminus of C and C-terminus of E flank the Rluc gene. ΔG values are in kcal/mol. (B) Kinetics of translation of WT DRrep and DRrep containing an inactive RdRP (RdRPmut). BHK cell monolayers were transfected with replicon RNAs and assayed for Rluc activity at 2, 4, 6, 8, 24, 48 and 72 hours post-transfection. Data shown are representative of 5 experiments. (C) Early translation events in DRrep, DRrep-HP3.4 and DRrep-HP11.9 replicon-transfected cells. Transfected cells were assayed for Rluc activity at 2, 4, 6 and 8 hours post-transfection. RLU are normalized for transfection efficiency. Data are averages of 5 experiments and error bars indicate SD. (D) Late translation events in DRrep-transfected cells. Transfected cells were assayed for Rluc activity at 2, 24, 48 and 72 hours post-transfection. RLU are normalized for transfection efficiency. Data are averages of 6 experiments, and error bars indicate SD. (E) Early and late translation events in infectious clone-transfected cells. Transfected cells were lysed at 2, 18 and 22 hours post-transfection and the cytoplasmic fraction was separated by SDS-PAGE. Immunoblots directed against the DENV NS1 protein and actin are representative of experiments. RLU, relative luciferase units. *, p < 0.003 relative to WT by Wilcoxon signed rank test.