FIG 3.

Characterization of resistant viruses. (A) Scheme of selection and validation of DENV-2 that is resistant to compound 1a inhibition. WT DENV-2 was used for resistance selection. The virus was cultured in A549 cells with increasing concentrations of compound 1a. The EC₅₀ for each selected isolate was measured at passage 16 (P16) by using qRT-PCR. The P16 viral RNA from each of the six independent selections was extracted for whole-genome sequencing. The extracted RNA was amplified by using SuperScript One-Step RT-PCR with Platinum Taq (Life Technologies). The RT-PCR products were sequenced. The identified mutations were introduced into a DENV-2 infectious cDNA clone to generate recombinant viruses. The resistance levels of recombinant viruses were evaluated by a viral titer reduction assay in A549 cells. (B) Resistance profile of the DENV-2 escape mutant. Mutations at position V63 of DENV-2 NS4B were consistently recovered from six independent resistant virus selections. DMSO (0.9%) was used as a negative control during resistance selection. Mixed mutations of V63 were obtained from selections I to IV. The sensitivities of the escape viruses to compound 1a (indicated by EC₅₀s) were compared to that of the WT virus. Fold resistance was calculated as the EC₅₀ for the resistant virus divided by the EC₅₀ for the WT virus. (C) Membrane topology and sequence alignment of the DENV NS4B protein. The identified V63 mutation is located in predicted transmembrane domain 2 (pTMD2) of the NS4B topology (22). An amino acid sequence alignment of the pTMD2 regions (residues 60 to 83) of different flavivirus NS4B proteins is shown. The amino acid positions of NS4B are numbered according to DENV-2 numbering (GenBank accession number AY037116). The V63 mutation in DENV-2 NS4B is highlighted; the equivalent position in DENV-1 NS4B is I64, as indicated. (D) Characterization of DENV-2 NS4B V63 mutants. BHK-21 cells were transfected with 10 μg of WT or NS4B mutant genome-length RNA of DENV-2. The transfected cells were monitored for viral E protein expression by IFA at days 2 and 4 p.t. (Top) Anti-E monoclonal antibody 4G2 and Alexa Fluor 488 goat anti-mouse IgG were used as the primary and secondary antibodies, respectively. (Middle) Plaque morphology of recombinant WT and mutant viruses. (Bottom) Viral titers in the culture fluids on day 5 p.t. were quantitated by a plaque assay. (E) Summary of resistance of the recombinant DENV-2 V63 mutants. Six recombinant DENV-2 V63 mutants were prepared and tested against compound 1a. The EC₅₀s and fold resistance values are shown.