Fig. 4.

Specific recognition of the capped viral RNA is essential for virus replication. (A) In vitro 2′-O-MTase activities of WT NS5 and E111 mutants using WT and mutant RNA templates. Data are normalized to the activity of the WT NS5 MTase on WT viral 5′UTR RNA, which is set to 100% (corresponding to ∼4 nM ³H-methyl incorporated into the RNA; signal-to-background count ratios are approximately four- to fivefold). Each data point is the average for three replicates, and error bars show the standard deviations. (B) Intracellular viral RNA levels detected by quantitative (q)RT-PCR. (C) Extracellular viral RNA levels in the supernatants detected by qRT-PCR. (D) Virus titers based on the plaque assay shown in E. (E) Plaque assay for WT and mutants at 24, 72, and 120 h posttransfection (hpt). The limits of detection for DENV genomic RNA by qRT-PCR and plaque assays are 100 copies and 1 infectious virus particle, respectively.