Abstract
Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.
Highlights
Dengue virus (DENV) is a mosquito-borne flavivirus, and infection with any of its four serotypes (DENV1-4) can result in inapparent infection, classic dengue fever, or dengue hemorrhagic fever/dengue shock syndrome–severe manifestations characterized by vascular leak that can lead to shock and death [1]
The upstream pathway triggered by non-structural protein 1 (NS1), as well as the molecular determinants of NS1 required for this phenomenon, remain obscure
N207-glycosylation and endocytosis of flavivirus NS1 are required for endothelial hyperpermeability based vaccine approaches that pose a minimal risk of antibody-dependent enhancement
Summary
Dengue virus (DENV) is a mosquito-borne flavivirus, and infection with any of its four serotypes (DENV1-4) can result in inapparent infection, classic dengue fever, or dengue hemorrhagic fever/dengue shock syndrome–severe manifestations characterized by vascular leak that can lead to shock and death [1]. We recently described a novel cell-intrinsic role for DENV NS1 in increasing permeability of human endothelial cell monolayers in vitro and systemic vascular leak in vivo via disruption of components of the endothelial glycocalyx-like layer (EGL) [8,9,10]. This EGL pathway is distinct from the cytokine-mediated pathway involving activation of peripheral blood mononuclear cells (PBMCs) previously described [10, 11]. The upstream pathway initiated by NS1, resulting in cathepsin L activation, EGL degradation, and vascular leak, as well as the molecular determinants of NS1 required for inducing endothelial hyperpermeability, have not yet been identified
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