Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E

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Abstract
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. Dengue virus (DENV) is a mosquito-transmitted virus that infects humans and has become a global emerging infectious disease threat. Four serotypes of DENV exist, and the most severe cases are associated with secondary infection with a different virus serotype. Clinical deterioration is characterized by bleeding and selective vascular leakage from endothelium in specific tissue sites. An increased understanding of how DENV proteins contribute to this phenotype is vital to developing novel vaccines and identifying individuals at risk for severe disease. DENV nonstructural protein-1 (NS1) is one such protein: during infection, it is secreted and accumulates in the supernatant and on the surface of cells. In this study, we demonstrate that soluble DENV NS1 attaches to subsets of cells, including some but not all endothelial cells, primarily via an interaction with specific glycosaminoglycans (heparan sulfate and chondroitin sulfate E). This was confirmed in tissue binding studies as DENV NS1 bound to lung and liver but not intestine or brain endothelium. Our findings suggest that the selective vascular leakage that occurs in severe DENV infection may be related to the relative ability of endothelial cells in different tissues to bind soluble NS1 and to be targeted by cross-reactive anti-NS1 antibodies during secondary infection.

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