Figure 1.
Time- and dose-dependent DENV infection of primary microvascular endothelial HMVEC-d cells and the endothelial cell line HMEC-1.
HMEC-1 (A, C) and HMVEC-d cells (E) were infected with DENV-2 at a MOI 1. Viral infectivity was quantified at different times after infection by flow cytometry using an anti-DENV-2 specific antibody (A, E). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells by means of real-time RT-PCR at different times after infection (C). Alternatively, HMEC-1 (B, D) and HMVEC-d cells (F) were treated with medium only (mock) or infected with DENV at a MOI of 1, 2 or 4. Viral infectivity was quantified 24 h post infection by flow cytometry (B, F). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells at 24 h post infection by means of real-time RT-PCR (D). The means and standard deviations of three independent experiments are shown.
Figure 2.
Expression of surface molecules on HMEC-1 and HMVEC-d cells as determined by flow cytometry.
HMEC-1 and HMVEC-d cells were stained with antibodies against different DENV receptors or endothelial cell markers. The mean fluorescence intensities for DC-SIGN, L-SIGN, mannose receptor (MR), heparan sulfate (HS), vascular endothelial growth factor receptor (VEGFR)-2 and CD31 (PECAM-1) are shown (after background reduction in the presence of labeled secondary antibody only or corresponding FITC-/PE-conjugated isotypic control antibody).
Figure 3.
Anti-DENV-2 activity of sulfated GAGs.
HMEC-1 cells were infected with DENV-2 at a MOI 1. Viral infectivity was quantified 24 h after infection by flow cytometry using an anti-DENV-2 specific antibody. Heparin (A), heparan sulfate (B), chondroitin sulfate A (C) and dermatan sulfate (D) dose-dependently inhibited DENV-2 infection in HMEC-1 cells. Data represent the % of infected cells relative to the positive control (DENV-2 infected cells). The means and standard deviations of three independent experiments are shown.
Table 1.
Antiviral activity of heparin and heparan sulfate analogs against DENV-2 in HMEC-1 cells.
Figure 4.
Heparinase II treatment of HMEC-1 cells reduces DENV-2 infectivity
. HMEC-1 cells were treated with 10 U/mL of heparinase II (A). Cell surface expression of heparan sulfate was analyzed by flow cytometry and plotted against the number of events (counts). The dashed line represents untreated HMEC-1 cells, the full histogram indicates heparinase II-treated HMEC-1 cells. Background in the absence of primary antibody is shown by the full line. HMEC-1 cells were treated with different concentrations of heparinase II (0 to 10 U/mL) and subsequently infected with DENV-2 at a MOI 1 (B). Viral infectivity was quantified by flow cytometry using an anti-DENV-2 specific antibody. The means and standard deviations of three independent experiments are shown.
Table 2.
Chemical features of sulfated K5 derivatives.
Figure 5.
Dose-dependent antiviral activity of K5-OS(H) and K5-N,OS(H) in DENV-infected endothelial cells and MDDC.
HMEC-1 or HMVEC-d cells (A, B) and MDDC (C, D) were infected with DENV-2 at a MOI 1 in the presence of different concentrations of K5-OS(H) (A, C) or K5-N,OS(H) (B, D). Viral infectivity was quantified 24 h (endothelial cells) or 48 h (MDCC) post infection by flow cytometry using an anti-DENV-2 specific antibody. Data represent the % of infected cells relative to the virus control. The means and standard deviations of three independent experiments are shown.
Figure 6.
Virus yield reduction assay on BHK cells.
HMEC-1 cells were infected with DENV-2 (MOI 1) in the presence or absence of the compounds (300 nM). After 24 h, supernatant was collected and transmitted to a monolayer of BHK cells at serial 10-fold dilutions. Plaques were counted after 4 days. Left: A representative virus yield reduction assay of dilution 10−1 is shown: Cell control (A), virus control (B), heparin (C), K5-OS(H) (D), K5-N,OS(H) (E) and K5 (F). Right: The % cytopathic effect (CPE) relative to the virus control ± standard deviation is shown.
Table 3.
Antiviral activity and cytotoxicity of K5 derivatives in HMEC-1 cells.
Figure 7.
K5 derivatives inhibit the early steps of DENV infection in microvascular endothelial cells.
K5-OS(H) (A) and K5-N,OS(H) (B) (500 nM) were added prior to (–30 min), together with (0 min), or at different times post-infection. The compounds were also incubated with virus inoculum for 30 min at 37°C before infection (virion). Viral infectivity was quantified after 24 h by flow cytometry using an anti-DENV-2 specific antibody. The percentage of infected cells relative to the virus control and standard deviation of 3 independent experiments are shown. Effect of K5-OS(H) and K5-N,OS(H) on DENV-2 attachment (C). HMEC-1 cells were incubated during 2 h at 4°C with DENV-2 in the presence or absence of various concentrations of compound. Next, cells were washed and after 24 h infection was analyzed by flow cytometry. Effect of K5-OS(H) and K5-N,OS(H) on DENV-2 entry (D). DENV-2 was allowed to attach to HMEC-1 cells for 2 h at 4°C. Unbound virus was washed away, various concentrations of compounds were added and temperature was raised to 37°C for 2 h to allow virus entry. Cells were washed and after 24 h infection was analyzed by flow cytometry. The percentage of infected cells relative to the virus control and standard deviation of 3 independent experiments are shown. Immunofluorescent staining of DENV-2 attachment to HMEC-1 cells (E). HMEC-1 cells were incubated with DENV-2 in the presence or absence of K5 derivatives (1.5 µM) on ice. Next, the nuclei (blue) and DENV-2 particles (green) were stained. K5-OS(H) and K5-N,OS markedly reduced DENV-2 attachment to HMEC-1 cells. CC: cell control, VC: virus control.
Figure 8.
Surface plasmon resonance (SPR) analysis of the interaction between K5 derivatives and the DENV envelope protein.
Sensorgrams show the binding of anti-DENV E antibody (positive control) and isotype antibody (negative control) (A), K5 (B), K5-OS(H) (C) and K5-N,OS(H) (D) to immobilized DENV E domain III. The binding curves of 0 to 120 s show the association, whereas those of 120 to 300 s show the dissociation phase. The y axis indicates the resonance signal as shown in resonance units (RU). Binding of DENV E domain III to immobilized heparin in the presence of different concentrations of compound (E). Heparin, K5-OS(H) and K5-N,OS(H) dose-dependently inhibited the interaction of domain III with immobilized heparin, whereas K5 was ineffective. Shown are the RU (% of domain III binding to the heparin chip).