Mutational analysis of NS5 carboxy-terminal residues Y838 and R888 in DENV2 by reverse genetics. (A) 3D structure of a DENV3 NS5 monomer (PDB access code: 5CCV.A [Klema et al. 2016]) with an enlarged view of the NS5 carboxy-terminal region showing hydrogen bond formation between Tyrosine 838 and Arginine 888 through their side chains. Replication characteristics of DENV2 WT NS5 and NS5 mutants are shown in B–E. BHK-21 cells were electroporated with 10 µg of DENV2 WT or mutant infectious clone RNA and the replication kinetics was followed until 96 h post-transfection. The replication-deficient NS5 mutant GAA was included as negative control. (B) Real-time PCR quantification of intracellular viral RNA at the indicated timepoints. The dotted line represented the detection level for mock-transfected cells. (C) Real-time PCR quantification of extracellular viral RNA in the supernatants of the transfected cells at the indicated timepoints. The dotted line represented the detection level in the supernatant of mock-transfected cells. (D) Virus titer in the supernatants of transfected BHK-21 cells measured by plaque assay. (E) Pictures showing plaque morphologies of DENV2 WT and NS5 mutant viruses. The dilution at which plaques can be observed is indicated. The data in B–D are presented as average ± SD from two independent experiments. Differences in intracellular (B) and extracellular (C) viral RNA kinetics between groups were compared by two-way ANOVA with Bonferroni correction. Mean values of the virus titers (D) between WT and the respective mutants are compared by unpaired Student's t-test, and a P-value <0.05 was considered significant (*) P < 0.05, (**) P < 0.01, (****) P < 0.0001.
