Limits...
Sulfated Escherichia coli K5 polysaccharide derivatives inhibit dengue virus infection of human microvascular endothelial cells by interacting with the viral envelope protein E domain III.

Vervaeke P, Alen M, Noppen S, Schols D, Oreste P, Liekens S - PLoS ONE (2013)

Bottom Line: Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface.Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.

ABSTRACT
Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface. However, heparan sulfate proteoglycans (HSPGs) were highly expressed on these cells and pre-treatment of HMEC-1 cells with heparinase II or with glycosaminoglycans reduced DENV infectivity up to 90%, suggesting that DENV uses HSPGs as attachment receptor on microvascular endothelial cells. Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range. The highly sulfated K5-OS(H) and K5-N,OS(H) inhibited virus attachment and subsequent entry into microvascular endothelial cells by interacting with the viral envelope (E) protein, as shown by surface plasmon resonance (SPR) analysis using the receptor-binding domain III of the E protein.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3755990&req=5

pone-0074035-g006: 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.

Mentions: K5 and K5-NS did not inhibit DENV-2 infection even at the highest concentration tested (3 µM), while K5-OS(L) and K5-N,OS(L) only inhibited DENV-2 infection by 42 and 19%, respectively, when administered at 3 µM, but lost activity at 5-fold lower concentrations. Instead the highly sulfated K5-OS(H) and K5-N,OS(H) proved to be very active compounds, which dose-dependently inhibited DENV-2 infection in the nanomolar range, with an EC50 value of 113±80 and 111±50 nM, respectively (Fig. 5, Table 3). The antiviral activity of the K5 derivatives was not due to cytotoxic effects (CC50 > 10 µM), resulting in a favorable selectivity index (SI) of 176 and 113 for K5-OS(H) and K5-N,OS(H), respectively (Table 3). Both compounds suppressed DENV-2 replication (measured by RT-PCR) with an EC50 of 168±88 and 201±57 nM, respectively (data not shown). To study the effect of sulfated K5 derivatives on infectious virus production, a virus yield reduction assay was performed in BHK cells using supernatant from HMEC-1 cells, infected with DENV in the presence (300 nM) or absence of the compounds. As expected, heparin (97%), K5-OS(H) (94%) and K5-N,OS(H) (91%), but not K5 (7%) significantly reduced the formation of plaques induced by infectious virus released from HMEC-1 cells (Fig. 6).


Sulfated Escherichia coli K5 polysaccharide derivatives inhibit dengue virus infection of human microvascular endothelial cells by interacting with the viral envelope protein E domain III.

Vervaeke P, Alen M, Noppen S, Schols D, Oreste P, Liekens S - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3755990&req=5

pone-0074035-g006: 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.
Mentions: K5 and K5-NS did not inhibit DENV-2 infection even at the highest concentration tested (3 µM), while K5-OS(L) and K5-N,OS(L) only inhibited DENV-2 infection by 42 and 19%, respectively, when administered at 3 µM, but lost activity at 5-fold lower concentrations. Instead the highly sulfated K5-OS(H) and K5-N,OS(H) proved to be very active compounds, which dose-dependently inhibited DENV-2 infection in the nanomolar range, with an EC50 value of 113±80 and 111±50 nM, respectively (Fig. 5, Table 3). The antiviral activity of the K5 derivatives was not due to cytotoxic effects (CC50 > 10 µM), resulting in a favorable selectivity index (SI) of 176 and 113 for K5-OS(H) and K5-N,OS(H), respectively (Table 3). Both compounds suppressed DENV-2 replication (measured by RT-PCR) with an EC50 of 168±88 and 201±57 nM, respectively (data not shown). To study the effect of sulfated K5 derivatives on infectious virus production, a virus yield reduction assay was performed in BHK cells using supernatant from HMEC-1 cells, infected with DENV in the presence (300 nM) or absence of the compounds. As expected, heparin (97%), K5-OS(H) (94%) and K5-N,OS(H) (91%), but not K5 (7%) significantly reduced the formation of plaques induced by infectious virus released from HMEC-1 cells (Fig. 6).

Bottom Line: Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface.Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.

ABSTRACT
Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface. However, heparan sulfate proteoglycans (HSPGs) were highly expressed on these cells and pre-treatment of HMEC-1 cells with heparinase II or with glycosaminoglycans reduced DENV infectivity up to 90%, suggesting that DENV uses HSPGs as attachment receptor on microvascular endothelial cells. Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range. The highly sulfated K5-OS(H) and K5-N,OS(H) inhibited virus attachment and subsequent entry into microvascular endothelial cells by interacting with the viral envelope (E) protein, as shown by surface plasmon resonance (SPR) analysis using the receptor-binding domain III of the E protein.

Show MeSH
Related in: MedlinePlus