Date Published: November 30, 2007
Publisher: Public Library of Science
Author(s): Panisadee Avirutnan, Lijuan Zhang, Nuntaya Punyadee, Ananya Manuyakorn, Chunya Puttikhunt, Watchara Kasinrerk, Prida Malasit, John P Atkinson, Michael S Diamond, Michael J Buchmeier.
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 hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe and potentially fatal complications of infection by dengue virus (DENV), a mosquito-borne RNA virus of the Flaviviridae family. Globally, DENV infects 25 to 100 million people per year, but the life-threatening complications primarily occur in school-age children . Four serotypes of DENV exist, and DHF/DSS is commonly associated with secondary infection with a different virus serotype [2,3]. In the most severe cases, clinical deterioration is characterized by a rapid decline after several days of continuous high fever, thrombocytopenia, and selective vascular leakage at serosal sites . The vascular leakage syndrome results in hemoconcentration, pleural effusions and ascites, and hypotension . An effective strategy for disease prevention or treatment is currently lacking.
Flavivirus NS1 is a secreted nonstructural glycoprotein that lacks a membrane-spanning region yet becomes cell surface–associated [31,42]. As NS1 attachment to the cell surface may be important for immune recognition [28,44] and immune evasion , we set out to identify the mechanism by which soluble NS1 binds to the surface of cells. Although it has been suggested that DENV NS1 attaches to the plasma membrane via a GPI anchor , this is unlikely to explain how DENV NS1 binds to uninfected cells. To assess this issue in greater detail, we developed a flow cytometric–based NS1 cell surface binding assay. We purified soluble DENV NS1 from the supernatants of baby hamster kidney (BHK) cells that stably express a DENV-2 subgenomic replicon (BHK DENV-2 Rep cells ). These cells produce high levels of intracellular (Figure 1A) and soluble NS1 as determined by a capture ELISA (3.6 ± 0.2 μg/ml of supernatant) and western blot (unpublished data). Elution profiles of purified NS1 after immunoaffinity or ion exchange chromatography are shown (Figure 1B and 1C, respectively). The yield of purified NS1 from BHK DENV-2 Rep cells (Figure 1D) was similar to that previously obtained from DENV-2-infected cell supernatants  and retained immunoreactivity as judged by western blot (Figures 1E and S1) or ELISA with a panel of conformationally sensitive anti-NS1 mAbs (unpublished data).
In this study, we demonstrate that soluble DENV NS1 binds to a subset of uninfected cells via interactions with GAG, primarily HS and CS-E. In cell culture, NS1 bound strongly to epithelial cells and fibroblasts, and weakly, if at all, to freshly isolated human peripheral blood leukocytes. Substantial variability was observed in NS1 binding to cultured endothelial cells and endothelium in situ. Experiments with the sulfation inhibitor sodium chlorate established that highly sulfated forms of GAG are required for optimal binding of soluble DENV NS1. Finally, our experiments suggest that NS1 on the surface of DENV-infected cells is linked primarily by a distinct, GAG-independent mechanism.