Fig 7. Role of Cter₁₈ NS5 examined in a DENV2 infectious clone.

(A-D) BHK-21 cells were electroporated with 10 μg of genomic-length RNA of WT DENV2 and mutants; supernatants and infected cells were harvested daily and consecutively for 5 days. Supernatants were used to (A and B) check for plaque production on BHK-21 cells and to (C) determine absolute copy numbers of extracellular viral RNA by real-time RT-PCR. Absolute copy numbers of viral RNA in log scale per ml of supernatant used for real-time RT-PCR was plotted; data are shown as the mean ± SD from two independent experiments. Data was normalised by deriving the absolute copy numbers from the mean values of the standards used in the two independent experiments. (D) RNA was extracted from infected cells and absolute copy numbers of intracellular viral RNA was determined by real-time RT-PCR. Absolute copy numbers of viral RNA per μg of RNA used for real-time RT-PCR was plotted; data are shown as the mean ± SD from two dependent experiments. The mutants were examined in two separate experiments. K887A/R888A, R890A/R891A and K330A were done in one set of experiment (n = 2) whereas R888A, R888E, R888K and P884T were done in a separate experiment (n = 2). WT was included in both experiment and only one set of WT data was shown here as representative of two independent experiments.