Inhibition of dengue virus entry into target cells using synthetic antiviral peptides

Abstract

Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.

Keywords: Antiviral peptides; Dengue virus; Domain III; Envelope protein; Inhibitory Peptides; Viral entry.

Figure 1

Toxicity of inhibitory peptides in vitro. MTS assay for cell viability was performed with increasing concentration of different peptides at different time courses 24, 48, and 72 hours. Results ranged from no evidence of toxicity to less than 10% for different peptides when compared to untreated control. (A) DET1, (B) DET2, (C) DET3, and (D) DET4. Results are expressed as mean from a representative experiment performed in triplicate.

Figure 2

Evaluation the antiviral activity of the designed peptides by plaque formation assay. The antiviral activity was tested by incubation LLC-MK2 target cells with a mixture of approximately 200 PFU of DENV-2 and 500 µM of each peptide. (A) Histogram shows that DET2 and DET4 are able to inhibit infection by DENV after normalized to untreated control. Results are expressed as mean ± SD from a representative experiment performed in triplicate. Asterisk denotes statistically significant differences from the untreated control (One-way ANOVA with Dunnett's post-test, P < 0.0001). (B) Plaque formation assay shows the reduction of plaque generation.

Figure 3

Evaluation the antiviral activity of the designed peptides by RT-qPCR. Viral RNA levels were quantified by RT-qPCR after pre-incubation with antiviral peptides. Data is expressed as relative fold expression compared to untreated control, which was defined as 1.0 fold, and then normalized to β-actin reference gene. Results showed that DET2 and DET4 significantly reduced DENV viral RNA load. The reduction level was 0.29 fold ± 0.16 (28.6% ± 16.3), and 0.81 fold ± 0.07 (81.0% ± 7.0) for DET2 and DET4 respectively. Results are expressed as mean ± SD from a representative experiment performed in quadruple experiments. Asterisk denotes statistically significant differences from the untreated control (One-way ANOVA with Dunnett's post-test, P < 0.0001).

Figure 4

Evaluation the antiviral activity of the designed peptides by Western blot. (A) Protein separation by SDS-PAGE. (B) Membrane incubated with normal human serum shows no bands. (C) Membrane incubated with dengue infected human serum develops bands for NS1, E, and prM proteins. (D) Relative quantification of E protein compared to untreated control, which was defined as 1.0 fold, and then normalized to β-actin reference protein. DET2 and DET4 significantly reduced the E protein (0.59 fold ± 0.11, and 0.78 fold ± 0.12 respectively). Results are expressed as mean ± SD from triplicate experiments. Asterisk denotes statistically significant differences from the untreated control (One-way ANOVA with Dunnett's post-test, P < 0.0001).

Figure 5

Dose-response curves for DET2 and DET4. The figure shows the effect of increasing concentrations of the DET2 and DET4 peptides against DENV-2. The DET2 peptide showed a maximum inhibitory activity against DENV-2 of 41.5% ± 20.0 at 200 µM (One-way ANOVA with Dunnett's post-test, P = 0.0065). The DET4 peptide showed a maximum inhibitory activity against DENV-2 of 84.6% ± 5.6 at 500 µM (One-way ANOVA with Dunnett's post-test, P < 0.0001). Results are expressed as mean ± SD from a representative experiment performed in triplicate. Asterisk denotes statistically significant differences from the untreated control.

Figure 6

Evaluation the post-infection antiviral activity of DET4 peptide. The ability of the peptide DET4 to inhibit DENV-2 replication after the virus entry into the target cells was determined by quantification of intracellular viral RNA load using RT-qPCR analysis. Peptides were added to the cells 24 hours post-infection. Then the viral RNA load in peptide treated cells was compared to the untreated control and was normalized to the reference gene (β-actin). Data is expressed as relative percent to untreated control, which was defined as 100%. Peptide DET4 showed no significant inhibitory effect against DENV-2 when added to the infected cells post-infection indicating that the peptide is active through the attachment and entry stage but not viral multiplication. Results are expressed as mean ± SD from a representative experiment performed in triplicate (One-way ANOVA with Dunnett's post-test, P = 0.7697, ns, denotes statistically non-significant differences from the untreated control).

Figure 7

Study the viral surface changes caused by peptide attachment using TEM. DENV was prepared with or without incubation with antiviral peptides before staining with PTA negative stain to visualize by TEM. (A and B) Untreated DENV as a control. (C) DENV treated with DET2 (D) DENV treated with DET4. Scale bars indicate 100 nm.