Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate

The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 x 10-9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.     

Primary attachment of murine leukaemia virus vector mediated by particle-associated heparan sulfate proteoglycan

Target cell entry of murine leukaemia virus vectors proceeds via primary attachment, independent of the viral envelope protein and subsequent envelope-receptor interaction. Although much attention has been paid to modifying the latter for target cell specificity, the initial binding interaction has been overlooked, despite its opposing involvement both in providing the virus available for receptor binding and in depleting free virus. As a first step towards modifying primary attachment, both to provide specificity and to enhance vector availability, we sought to determine the nature of this interaction. Following an initial screen of GAGs (glycosaminoglycans) for their ability to inhibit virus binding and transduction, we have shown that production of virus from cells in which GAG sulfation is inhibited, or treatment of virus with heparinase III, reduces both particle attachment and infection. Detection in purified virus preparations of a neo-epitope generated by heparinase III confirmed the presence of virus-associated HSPG [HS (heparan sulfate) proteoglycan], acquired from the producer cell. We propose that host-acquired cell-surface HSPG (potentially including syndecan-2) provides a means of virus attachment to target cells that precedes specific receptor interaction and membrane fusion. Inhibition of HS biosynthesis may provide a sufficiently reduced background of primary binding such that novel mechanisms of attachment, ideally with appropriate target cell specificity, can be introduced.     

Herpes simplex virus type 1-induced hemagglutination: glycoprotein C mediates virus binding to erythrocyte surface heparan sulfate.

We recently reported that herpes simplex virus type 1 (HSV-1) can cause agglutination of murine erythrocytes (E. Trybala, Z. Larski, and J. Wisniewski, Arch. Virol. 113:89-94, 1990). We now demonstrate that the mechanism of this hemagglutination is glycoprotein C-mediated binding of virus to heparan sulfate moieties at the surface of erythrocytes. Hemagglutination was found to be a common property of all gC-expressing laboratory strains and clinical isolates of HSV-1 tested. Mutants of HSV-1 deficient in glycoprotein C caused no specific hemagglutination, whereas their derivatives transfected with a functional gC-1 gene, thus reconstituting gC expression, regained full hemagglutinating activity. Hemagglutination activity was inhibited by antibodies against gC-1 but not by antibodies with specificity for glycoproteins gB, gD, or gE or by murine antiserum raised against the MP strain of HSV-1, which is gC deficient. Finally, purified gC-1 protein, like whole HSV-1 virions, showed high hemagglutinating activity which was inhibited by heparan sulfate and/or heparin and was completely prevented by pretreatment of erythrocytes with heparitinase, providing evidence that gC-1 mediates hemagglutination by binding to heparan sulfate at the cell surface. Thus, HSV-1-induced hemagglutination is gC-1 dependent and resembles the recently proposed mechanism by which HSV-1 attaches to surface heparans on susceptible cells, providing a simple model for initial events in the virus-cell interaction.     

The binding of the circumsporozoite protein to cell surface heparan sulfate proteoglycans is required for plasmodium sporozoite attachment to target cells

The major surface protein of malaria sporozoites, the circumsporozoite protein, binds to heparan sulfate proteoglycans on the surface of hepatocytes. It has been proposed that this binding event is responsible for the rapid and specific localization of sporozoites to the liver after their injection into the skin by an infected anopheline mosquito. Previous in vitro studies performed under static conditions have failed to demonstrate a significant role for heparan sulfate proteoglycans during sporozoite invasion of cells. We performed sporozoite attachment and invasion assays under more dynamic conditions and found a dramatic decrease in sporozoite attachment to cells in the presence of heparin. In contrast to its effect on attachment, heparin does not appear to have an effect on sporozoite invasion of cells. When substituted heparins were used as competitive inhibitors of sporozoite attachment, we found that sulfation of the glycosaminoglycan chains at both the N- and O-positions was important for sporozoite adhesion to cells. We conclude that the binding of the circumsporozoite protein to hepatic heparan sulfate proteoglycans is likely to function during sporozoite attachment in the liver and that this adhesion event depends on the sulfated glycosaminoglycan chains of the proteoglycans.     

Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.     

Role of the N- and C-terminal domains in binding of apolipoprotein E isoforms to heparan sulfate and dermatan sulfate: a surface plasmon resonance study

The ability of apolipoprotein E (apoE) to bind to cell-surface glycosaminoglycans (GAGs) is important for lipoprotein remnant catabolism. Using surface plasmon resonance, we previously showed that the binding of apoE to heparin is a two-step process; the initial binding involves fast electrostatic interaction, followed by a slower hydrophobic interaction. Here we examined the contributions of the N- and C-terminal domains to each step of the binding of apoE isoforms to heparan sulfate (HS) and dermatan sulfate (DS). ApoE3 bound to less sulfated HS and DS with a decreased favorable free energy of binding in the first step compared to heparin, indicating that the degree of sulfation has a major effect on the electrostatic interaction of GAGs with apoE. Mutation of a key Lys residue in the N-terminal heparin binding site of apoE significantly affected this electrostatic interaction. Progressive truncation of the C-terminal alpha-helical regions which favors the monomeric form of apoE3 greatly weakened the ability of apoE3 to bind to HS, with a much reduced favorable free energy of binding of the first step, suggesting that the C-terminal domain contributes to the GAG binding of apoE by the oligomerization effect. In agreement with this, dimerization of the apoE3 N-terminal fragment via disulfide linkage restored the electrostatic interaction of apoE with HS. Significantly, apoE4 exhibited much stronger binding to HS and DS than apoE2 or apoE3 in both lipid-free and lipidated states, perhaps resulting from enhanced electrostatic interaction through the N-terminal domain. This isoform difference in GAG binding of apoE may be physiologically significant such as in the retention of apoE-containing lipoproteins in the arterial wall.     

Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.     

Cathepsin X binds to cell surface heparan sulfate proteoglycans

Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin-Sepharose. Conformational changes were observed to accompany heparin-cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the kcat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.     

New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, (13)C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the alpha-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.     

Biology of cell surface heparan sulfate proteoglycans

The central question in cell biology is how cells detect, interact and respond to extracellular matrix. The cell surface molecules, which mediate this recognition, consist of a lipophilic membrane domain and an ectodomain binding matrix materials. One group of this kind of molecules is the cell surface heparan sulfate proteoglycans (HSPG). This review summarizes recent information obtained on the cell surface PG of mouse mammary epithelial cells. The glycosaminoglycan containing ectodomain of this PG binds with high affinity Type I, III and V collagen fibrils and the C-terminal heparin binding domain of fibronectin. The PG is mobile on the cell surface, but can be immobilised by ligand binding. At the same time the PG associates with cytoskeleton and links the epithelial cytoskeleton to extracellular matrix. Thus the PG can mediate the changes in the matrix into changes in cellular behaviour, often seen during the regulation of cell shape, proliferation and differentiation. The cell surface PG is also released from the cell surface by cleaving the matrix-binding ectodomain from the membrane domain. Because of the binding properties of the ectodomain, this shedding may provide a means by which epithelial cells loosen their association with the matrix and with other cells, e.g., during normal epithelial development and the invasion of carcinomas.     

Helicobacter pylori vacuolating cytotoxin binding to a putative cell surface receptor, heparan sulfate, studied by surface plasmon resonance

The Helicobacter pylori vacuolating cytotoxin or VacA toxin is a major virulence factor in H. pylori infection and type B gastritis. We predicted heparin/heparan sulfate (H/HS) binding properties of the 58-kDa subunit of VacA cytotoxin using bioinformatics tools and showed this by surface plasmon resonance (SPR)-based biosensor studies. Putative H/HS binding peptides were synthesized and binding to HS was shown by SPR in the absence or presence of trifluoroethanol. We found that a recombinant cytotoxin VacA polypeptide binds to surface-immobilized HS and propose that HS might be a receptor/co-receptor for H. pylori VacA cytotoxin.     

Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans

It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease.     

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.     

A conformational change in heparan sulfate 3-O-sulfotransferase-1 is induced by binding to heparan sulfate

The 3-O-sulfation of glucosamine by heparan sulfate 3-O-sulfotransferase-1 (3-OST-1) is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). In this paper, we present evidence of a conformational change that occurs in 3-OST-1 upon binding to heparan sulfate. The intrinsic fluorescence of 3-OST-1 was increased in the presence of HS, suggesting a conformational change. This apparent conformational change was further investigated using differential chemical modification of 3-OST-1 to measure the solvent accessibility of the lysine residues. 3-OST-1 was treated with acetic anhydride in either the presence or absence of HS using both acetic anhydride and hexadeuterioacetic anhydride under nondenaturing and denaturing conditions, respectively. The relative reactivity of the lysine residues to acetylation and [2H] acetylation in the presence or absence of HS was analyzed by measuring the ratio of acetylated and deuterioacetylated peptides using matrix-assisted laser desorption ionization mass spectrometry. The solvent accessibilities of the lysine residues were altered differentially depending on their location. In particular, we observed a group of lysine residues in the C-terminus of 3-OST-1 that become more solvent accessible when 3-OST-1 binds to HS. This observation indicates that a conformational change could be occurring during substrate binding. A truncated mutant of 3-OST-1 that lacked this C-terminal region was expressed and found to exhibit a 200-fold reduction in sulfotransferase activity. The results from this study will contribute to our understanding of the interactions between 3-OSTs and HS.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans.

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

Human papillomavirus infection requires cell surface heparan sulfate

Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.     

High molecular-weight heparan sulfate from the cell surface

Heparan sulfate fragments with molecular weight of 135,000 (as determined by equilibrium sedimentation analysis) were isolated from the trypsinate of Chinese hamster cells (line CHO) grown in culture. Evidence is presented which suggests that the intracellular heparan sulfate species with molecular weight of 10,000 to 20,000 were degradation products of the larger species. We propose that the native cell-surface heparan sulfate, in its physiological location, could serve as a nonspecific “screen” to the exposure of specific, topographically adjacent, cell-surface sites.     

Echoviruses bind heparan sulfate at the cell surface

Some echoviruses (EV) that bind decay-accelerating factor (DAF) also bind cells of human and murine origins in a DAF-independent manner. Pretreatment of cells with heparinase 1 or heparin blocks the binding of radiolabeled virus to the cell surface, and heparin prevents infection of rhabdomyosarcoma cells by certain EV, including several low-passage clinical isolates of EV 6 and some EV that do not bind DAF. These studies suggest that heparan sulfate may be of in vivo relevance as an attachment molecule for EV.     

Hepatitis B virus surface antigen binds to apolipoprotein H.

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection.