Figures and data
Generation of DENV2 fusion loop mutants via directed evolution. A) Alignment of Top: Dengue virus fusion loops; Bottom: Mosquito-borne flavivirus fusion loops, including Yellow Fever virus (YFV), Zika virus (ZIKV), West Nile virus (WNV), Kunjin virus (KUNV), Murray Valley Encephalitis virus (MVEV), Japanese Encephalitis virus (JEV), Usutu virus (USUV), and Saint Louis Encephalitis virus (SLEV). Amino acids are colored by functional groups: negatively charged (red), positively charged (blue), nonpolar (yellow), polar (green), aromatic (pink), and sulfide (dark red). B) Schematic of directed evolution procedure. Saturation mutagenesis libraries were used to produce viral libraries, which were passaged three times in either C6/36 or Vero 81 cells. At the end of the selection, viral genomes were isolated and mutations were identified by high-throughput sequencing. C) Left: Bubble plot of the sequences identified from either the unselected or selected (passage 3) C6/36 DENV libraries. Right: Pie chart of the sequences from passage 3 C6/36 DENV libraries. D) Structure of the DENV envelope with the fusion loop mutations highlighted in red. E) Sequences of the fusion loop and furin cleavage site of DENV2, D2-FL, and D2-FLM.
Biological and physical properties of mature DENV2 fusion loop mutants. A) Multistep growth curves (MOI = 0.05-0.1) of DV2-WT, D2-FL, and D2-FLM on C6/36 cells (left) or Vero 81 cells (right). B) Thermostability assay on DV2-WT, D2-FL, and D2-FLM. C) Western blot of virions, blotted against Envelope and prM proteins. The prM:E ratio was determined and normalized to the DV2-WT ratio. Averages of 3 biological replicates are shown. Two-way ANOVA was used for statistical comparison of growth curves and thermostability: ns = not significant; * < 0.05; ** < 0.005; *** < 0.0005.
Fusion loop mutant is insensitive to fusion loop mAbs, the major target for cross-reactive Abs in NHPs. A) Left: FRNT50 values for neutralization of DV2-WT, D2-FL, and D2-FLM with mAbs against the FL (1M7, 1N5, 1L6, 4G2). All Abs were tested in at least n=3 independent experiments, except 1N5 due to limited Ab. Right: Average neutralization curves for neutralization of DV2-WT, D2-FL, and D2-FLM with mAbs against the DENV2 fusion loop. B) FRNT50 values for neutralization of DV2-WT and D2-FLM with mAbs against DENV2 prM (2H2, 1E16, 5M22), EDI (3F9), EDE (C10, B7), and EDIII (2D22). All Abs were tested in at least n=3 independent experiments. C) Neutralization of DV2-WT, D2-FL, and D2-FLM with sera from NHPs infected with either DENV4 or DENV2. FRNT50s were compared using Student’s t-test. Significant symbols are as follows: *, P < 0.05; **, P< 0.005; ***, P < 0.0005; ****, P < 0.00005. The data are graphed as means ± standard deviations.
