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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.

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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.
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pone-0074035-g001: 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.

Mentions: We investigated DENV infection and replication in microvascular endothelial cells by means of flow cytometry and RT-PCR at different time points post-infection. When HMEC-1 cells were infected with DENV-2 at a MOI of 1, 11% of the cells were infected at 24 h post-infection (Fig. 1A). This number could be increased up to 49% when a MOI of 4 was used (Fig. 1B). Histograms representing flow cytometric analyses are shown in Fig. S1. The infected cells were susceptible to DENV replication as shown by the increase in viral load 24-48 h post-infection (Fig. 1C, D).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755990&req=5

pone-0074035-g001: 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.
Mentions: We investigated DENV infection and replication in microvascular endothelial cells by means of flow cytometry and RT-PCR at different time points post-infection. When HMEC-1 cells were infected with DENV-2 at a MOI of 1, 11% of the cells were infected at 24 h post-infection (Fig. 1A). This number could be increased up to 49% when a MOI of 4 was used (Fig. 1B). Histograms representing flow cytometric analyses are shown in Fig. S1. The infected cells were susceptible to DENV replication as shown by the increase in viral load 24-48 h post-infection (Fig. 1C, D).

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