Table 1.
Sequences and IC50 values of peptides.
Figure 1.
Locations of predicted peptides on the DENV-2 E protein primary sequence.
(A) The DENV-2 E protein is shown linearly from N to C terminus. The three domains are color coded above, domain I is shown in red, domain II is yellow, and domain III is in blue according to [46]. The calculated RAPDF scores of a sliding window of 20 amino acid peptide sequences are shown graphically by vertical black lines. Four major high binding regions are predicted, from amino acids 31–70, 121–160, 241–270, and 341–380, respectively, corresponding to the locations of peptides 1OAN1, 1OAN2, 1OAN3, and 1OAN4. The locations of the other optimized peptides are indicated by three horizontal black lines, amino acids 96–114 is DN80opt, 205–223 is DN81opt, and 205–232 is DN57opt. (B) Structure of dimeric dengue E protein in the pre-fusion conformation showing locations of inhibitory peptides. Top, side, and bottom views are shown. Structures are color coded as above. Black and grey residues show the positions of the 1OAN1 and DN57opt peptides respectively. (C) Structure of monomeric dengue E protein in the low pH post-fusion conformation. Inner (interacting) and outer surfaces are shown.
Figure 2.
Inhibition of DENV-2 in vitro.
Increasing concentrations of optimized inhibitor peptides and corresponding scrambled peptides of identical composition were tested against DENV-2 in a focus forming unit reduction assay. (A) Optimized peptides (B) DN57opt and corresponding scrambled peptide of identical composition (C) Novel peptides (D) 1OAN1 and corresponding scrambled peptide of identical composition. Error bars are ±sem.
Figure 3.
Inhibitory peptide toxicity in vitro.
Increasing concentrations of peptides were tested in an MTT mitochondrial reductase activity assay. Error bars are ± sd. (A) DN57opt (B) Scrambled version of DN57opt (C) 1OAN1 (D) Scrambled version of 1OAN1. * denotes a statistically significant difference from the no peptide control.
Figure 4.
Purified and concentrated virus was prepared with or without incubation with peptides and then flash frozen for imaging. Panels show (A) virus only, (B) virus incubated with DN57opt, (C) virus incubated with 1OAN1. Scale bars indicate 100 nm.
Figure 5.
Peptide:E protein binding assay.
Biolayer interferometry was used to assay the binding of the peptides to truncated E Protein. The association and dissociation of increasing concentrations of truncated E protein to peptides DN57opt (A) and 1OAN1 (B) are shown. A buffer blank (PBS, 0.02% Tween-20, 0.1% BSA) containing no E protein was run for each peptide. The affinity of the peptides for the truncated E protein was calculated (DN57opt KD = 1.2×10−6±0.6×10−6 M (mean±sd), 1OAN1 KD = 4.5×10−7±2.0×10−7 M).
Figure 6.
Post-infection and post-binding peptide treatments.
Treatment of cells with increasing concentrations of (A) DN57opt and (B) 1OAN1 after DENV-2 has infected cells shows no significant inhibition. Treatment with (C) DN57opt or (D) 1OAN1 after DENV-2 has bound to LLCMK-2 cells at 4°C for one hour inhibits infection. Error bars are ±sem.
Figure 7.
Quantitative reverse transcriptase PCR virus:cell binding.
Virus pre-incubated with either DN57opt or 1OAN1 shows reduced binding to cells compared to control virus without peptide. Pre-incubation of virus with pooled human anti-dengue serum or heparan sulfate similarly shows reduced cell binding. * Indicates a significant difference (p<0.05) from all others by 1-way ANOVA followed by Tukey's posthoc test.