Peptide inhibitors of dengue virus and West Nile virus infectivity

Abstract

Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 microM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of <25 microM) and cross-inhibition of WNV fusion/infectivity (>99% inhibition at <25 microM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection.

Figure 1

Diagramatic structure of DENV envelope protein showing inhibitory peptide regions. Grey lines: dicysteine linkages. Black stick figures: N-glycosylation sites. Regions with significant Wimley-White interfacial hydrophobicity scale scores were predicted with MPeX (Boxed in left depiction; black in right depiction). Sequences of DENV peptides and the location of WNV homologs are indicated.

Figure 2

Plaque inhibition assay. (A) Preincubation of DENV with neutralizing antiserum reduces plaque number by 74%. (B) Preincubation of DENV with a non-inhibitory peptide shows no reduction in plaques. (C) Preincubation of DENV with one of the inhibitory peptides (DN59) shows a greater than 95% reduction in plaques.

Figure 3

Dose-response curves for WN53, WN83 and DN59 peptides. (A) Increasing concentrations of peptide WN53 produce a corresponding increase in WNV inhibition with an IC50 in the 10μ range. (B) Increasing concentrations of peptide WN83 produce a corresponding increase in WNV inhibition with an IC50 in the 10 μM range. (C) Increasing concentrations of peptide DN59 also produce a corresponding increase in DNV inhibition with an IC50 in the 10 μM range. All measurements were made in triplicate, with mean +/- SD shown.

Figure 6

Inhibitory effect of peptides WN53 and DN59 alone and in combination. WN53 and DN59 peptides were tested alone (■ DN59 alone, ● WN53 alone) or together (◆) with WNV and inhibitory activity at three concentrations was measured (mean of three trials +/- SD shown). DN59 and WN53 together have an effect intermediate between the two peptides alone.

Figure 4

Effect of DENV and WNV specific peptides against another virus. Peptides DN59, WN83 and WN53 at 100 μg/ml concentrations were tested for inhibitory activity against the alphavirus SINV in a similar plaque reduction assay. Results are shown as the mean of three trials +/- SEM. None of the peptides showed a statistically significant inhibitory effect against SINV (ANOVA, p = 0.705, with Dunnett's posthoc test).

Figure 5

Effect of scrambled peptide order on inhibitory function. Scrambled versions of peptides DN59 and WN83 at 100 μg/ml concentrations were tested for inhibitory effect against DENV and WNV, respectively. Scrambled versions of the peptides showed no inhibitory activity compared to the original DN59 and WN83 peptides.

Figure 7

Toxicity of inhibitory peptides in cell culture. MTT assays for cell viability were performed after 24 hr incubation of cells with 100 μg/ml of peptides WN83, WN53, and DN59 (mean from three experiments +/- SEM). No statistically significant differences in cell viability were observed (ANOVA p = 0.672, with Dunnett's posthoc test).