Evolution of a functionally intact but antigenically distinct DENV fusion loop
- Department of Molecular Microbiology and Immunology, Saint Louis University, United States
- Department of Epidemiology, University of North Carolina at Chapel Hill, United States
- Department of Microbiology, University of North Carolina at Chapel Hill, United States
Figures
Figure 1
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.
Figure 2
Biological and physical properties of mature DENV2 fusion loop mutants.
(A) Multistep growth curves [Multiplicity of infection (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. Equal volumes of supernatants from viral infected C6/36 cells at 5 days post-infection (dpi) (A) were loaded into each lane. The prM:E ratio was determined and normalized to the DV2-WT ratio. High exposure of D2-FLM (right) illustrates the relative abundance of prM protein to E protein. Averages of three biological replicates are shown. The data are graphed as means ± standard deviations (Error bars). Two-way ANOVA was used for statistical comparison of growth curves and thermostability: ns, not significant; *<0.05; **<0.005; ***<0.0005.
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Figure 2—source data 1
Raw gel images of the western blot shown in Figure 2C.
- https://cdn.elifesciences.org/articles/87555/elife-87555-fig2-data1-v1.zip
Figure 3
Fusion loop (FL) mutant is insensitive to FL monoclonal antibodies (mAbs), the major target for cross-reactive Abs in non-human primates (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 FL. (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. The data are graphed as means ± standard deviations (Error bars). (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.
Tables
Table 1
Summary of FRNT50 values of human convalescent serum and non-human primates (NHP) infection serum against DV2, D2-FL (not determined in human serum) and D2-FLM.
FRNT50 values were derived from an eight-point dilution curve of serum started from 1:40 with subsequent threefold dilutions. FRNT50 values were calculated/extrapolated from the neutralization curves. For human serum, the average FRNT50 values were shown from two independent experiments with technical replicates. For NHP serum, FRTN50 values were average of three independent experiments. ND, not determined. * denotes the Hill slopes of all the neutralization curve of them sample are <0.5 (low confidence of neutralization).
| Human # | Infected serotype | Collection tme | DV2 | D2-FL | D2-FLM |
|---|---|---|---|---|---|
| DS1500 | DENV1 | Convalescent serum | 1:75 | ND | 1:53 |
| DS2499 | 1:80 | ND | 1:52 | ||
| DS1136 | DENV3 | 1:133 | ND | 1:45 | |
| DS1160 | 1:86 | ND | 1:208 | ||
| DS0275 | DENV4 | 1:94 | ND | 1:94 | |
| DS2239 | 1:156 | ND | 1:42 | ||
| NHP # | Infected serotype | Collection time (dpi) | DV2 | D2-FL | D2-FLM |
| R628 | DENV4 | 30 | <1:40 | <1:40 | <1:40 |
| 60 | <1:40 | <1:40 | <1:40 | ||
| 90 | 1:42* | <1:40 | <1:40 | ||
| R737 | 30 | <1:40 | <1:40 | <1:40 | |
| 60 | <1:40 | <1:40 | <1:40 | ||
| 90 | <1:40 | <1:40 | <1:40 | ||
| R1132 | 30 | <1:40 | <1:40 | <1:40 | |
| 60 | <1:40 | <1:40 | <1:40 | ||
| 90 | <1:40 | <1:40 | <1:40 | ||
| R1160 | 30 | 1:58* | <1:40 | <1:40 | |
| 60 | 1:57* | 1 : 50* | <1:40 | ||
| 90 | <1:40 | 1 : 57* | 1:91* |
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Evolution of a functionally intact but antigenically distinct DENV fusion loop
eLife 12:RP87555.
https://doi.org/10.7554/eLife.87555.3
