Productive dengue virus infection of human endothelial cells is directed by heparan sulfate-containing proteoglycan receptors - PubMed (original) (raw)

Productive dengue virus infection of human endothelial cells is directed by heparan sulfate-containing proteoglycan receptors

Nadine Dalrymple et al. J Virol. 2011 Sep.

Abstract

Dengue virus causes leakage of the vascular endothelium, resulting in dengue hemorrhagic fever and dengue shock syndrome. The endothelial cell lining of the vasculature regulates capillary permeability and is altered by immune and chemokine responses which affect fluid barrier functions of the endothelium. Our findings indicate that human endothelial cells are highly susceptible to infection by dengue virus (type 4). We found that dengue virus productively infects ∼80% of primary human endothelial cells, resulting in the rapid release of ∼10(5) virions 1 day postinfection. Analysis of potential inhibitors of dengue virus entry demonstrated that antibodies and ligands to integrins and cellular receptors were unable to inhibit dengue virus infection of endothelial cells. In contrast, pretreating cells with heparin or heparan sulfate resulted in a 60 to 80% reduction in dengue virus-infected cells, and pretreatment of endothelial cells with heparinase III or protease reduced dengue infectivity by >80%. Dengue virus bound specifically to resin immobilized heparin, and binding was competitively inhibited by excess heparin but not other ligands. Collectively, these findings suggest that dengue virus specifically attaches to heparan sulfate-containing proteoglycan receptors on endothelial cells. Following attachment to human endothelial cell receptors, dengue virus causes a highly productive infection that has the potential to increase viral dissemination and viremia. This provides the potential for dengue virus-infected endothelial cells to directly alter barrier functions of the endothelium, contribute to enhancement of immune cell activation, and serve as potential targets of immune responses which play a central role in dengue pathogenesis.

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Figures

Fig. 1.

Productive infection of primary human endothelial cells by dengue virus. (A) Dengue virus type 4 (DV4) was adsorbed to primary human endothelial cells or Vero E6 cells at an MOI of 6 or mock infected for 1.5 h at room temperature. Inocula were removed, and cell monolayers washed with PBS three times. Cells were grown in complete medium for 24 h, the medium removed, and monolayers fixed with 100% ice-cold methanol for 10 min. Dengue virus antigen in infected cells was detected using DV4-specific HMAF antibody (1:4,000) followed by binding to a horseradish peroxidase-tagged secondary antibody (1:4,000) and immunoperoxidase staining using amino-ethyl carbazole (26, 28, 68). (B) Uninfected (U) or dengue virus-infected (I) endothelial or Vero E6 cells were treated as described for panel A, and cell lysates were collected 24 h postinfection. Proteins were separated by SDS-PAGE (10%) and Western blotted using anti-DV4 NS1 peptide (1:300), anti-DV4 capsid peptide (1:1,000), or anti-actin (1:1,000) antibodies. (C) Endothelial and Vero E6 cells were infected in duplicate as described for panel A with a constant amount of dengue virus (MOI of 6), washed with PBS, and incubated (37°C, 5% CO2) for various lengths of time. The titers of dengue virus present in supernatants of endothelial and Vero E6 cells (12, 24, 48, and 72 h postinfection) were determined on C6/36 cells using DV4 HMAF antibody and immunoperoxidase staining as described for panel A. Dengue virus antigen-containing infected-cell foci were quantitated and reported as focus forming units (FFU).

Fig. 2.

Effect of endothelial cell receptor blockade on dengue virus infection. Primary human endothelial cells were pretreated with antibodies (0.5 or 5 μg/ml) to the indicated cellular receptors or a control antibody to green fluorescent protein (GFP) in duplicate (1 h at 4°C), followed by species-specific secondary antibody (1:2,000, 1 h, 4°C) (26, 28, 68). Subsequently, 100 FFU of DV4 was adsorbed to endothelial cell monolayers (1 h), which were then washed 3 times with PBS and incubated for 24 h (37°C, 5% CO2). Dengue virus antigen-positive infected cells were quantitated as described for Fig. 1A using HMAF antibody and an immunoperoxidase staining assay. The number of dengue virus-infected cells following receptor blockade are reported as the percentage of infected cells treated with a control GFP antibody (100%).

Fig. 3.

Ligands inhibit endothelial cell infection by dengue virus. Dengue virus (150 FFUs) was prebound to potentially competitive ligands (1 and 100 μg/ml, 1 h) prior to infection of endothelial cells. Following adsorption, endothelial cell monolayers were fixed 24 h postinfection and the number of dengue virus-infected cells was determined by the DV4-HMAF antibody immunoperoxidase staining assay described for Fig. 1A. The number of endothelial cells infected in the presence of a specific ligand is presented as the percentage of infected cells observed in control BSA-treated samples (100%).

Fig. 4.

Heparin inhibits dengue virus infection of endothelial cells. Dengue virus (150 FFU) was prebound to increasing concentrations (0.1 to 100 μg/ml, 1 h) of heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate A, or BSA diluted in DMEM. Following adsorption of the DV4 samples onto endothelial cells, the monolayers were washed and incubated at 37°C for 24 h. The number of dengue virus-infected cells was determined by the DV4-HMAF antibody immunoperoxidase staining assay described for Fig. 1A. The number of endothelial cells infected in the presence of a specific ligand is presented as the percentage of infected cells observed in control BSA-treated endothelial cells (100%).

Fig. 5.

Enzymatic treatment of human endothelial cells. Endothelial cell monolayers were incubated with heparinase III (0.5 to 3 U/ml) (white bars) or chondroitinase (0.5 to 3 U/ml) (black bars) (A) or proteinase K (0.01 to 10 μg/ml, striped bars) (B) or were mock treated (gray bar; A, B) for 30 to 60 min at 37°C. Monolayers were washed with PBS, and dengue virus (150 FFU) was adsorbed to cells (1 h). Following adsorption, endothelial cell monolayers were washed and incubated at 37°C for 24 h. The number of dengue virus-infected cells was determined by the DV4-HMAF antibody immunoperoxidase staining assay described for Fig. 1A. The number of endothelial cells infected following enzymatic treatment is reported as the percentage of infected cells observed in mock-treated, no-enzyme-added controls (100%).

Fig. 6.

Dengue virus binds to immobilized heparin. Binding of dengue virus to heparin-Sepharose or unmodified Sepharose was used to determine whether dengue virus was capable of binding heparin. Dengue virus (8 × 108 FFU) was pretreated with 1 mg of soluble heparin, vitronectin, or sialic acid (in DMEM, 1 h, 4°C) in duplicate and subsequently incubated with heparin-Sepharose resin or Sepharose resin alone (1 h, 4°C with rotation). Resin was pelleted and washed 3 times with DMEM, and bound virus was eluted with DMEM containing 1.5 M NaCl. Eluates were diluted 3-fold with medium, and the titer of infectious DV4 bound to resin was determined on C6/36 cells as described for Fig. 1C. Dengue virus bound to resin in the presence of excess ligand is reported as the percentage of virus bound to heparin-conjugated Sepharose alone (100%).

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Productive dengue virus infection of human endothelial cells is directed by heparan sulfate-containing proteoglycan receptors - PubMed (original) (raw)