Date Published: July 29, 2019
Publisher: Public Library of Science
Author(s): Chunling Wang, Henry Puerta-Guardo, Scott B. Biering, Dustin R. Glasner, Edwina B. Tran, Mark Patana, Trent A. Gomberg, Carmel Malvar, Nicholas T. N. Lo, Diego A. Espinosa, Eva Harris, Sara Cherry.
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.
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 . West Nile virus (WNV) is a related flavivirus that causes encephalitis , and Zika virus (ZIKV), which recently emerged and generated large epidemics across the Americas, can cause Guillain-Barré syndrome in adults and microcephaly and other congenital birth defects in babies born to women infected during pregnancy [3–7]. The 10.7-kb RNA genome of flaviviruses encodes three structural and seven non-structural (NS) proteins, including NS1. 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) [8–10]. This EGL pathway is distinct from the cytokine-mediated pathway involving activation of peripheral blood mononuclear cells (PBMCs) previously described [10, 11]. The NS1-mediated degradation of the EGL involves activation of the protease cathepsin L, heparanase, and sialidases, which in turn disrupt the EGL. However, the upstream pathway initiated by NS1, resulting in cathepsin L activation, EGL degradation, and therefore vascular leak, as well as the molecular determinants of NS1 required for inducing endothelial hyperpermeability, have not yet been identified.
In this study, we demonstrate that the N207 glycosylation site of DENV NS1 is essential for inducing EGL degradation and hyperpermeability of human endothelial cells. We found that the DENV NS1-N207Q mutant binds to cells at similar levels as WT DENV NS1 but is retained on the cell surface, in contrast to WT DENV NS1, which is rapidly internalized. Additionally, internalization of WT DENV NS1 was dynamin- and clathrin-dependent, but independent of caveolin. Further, clathrin-mediated endocytosis of WT DENV, WNV, and ZIKV NS1 was required for endothelial hyperpermeability and EGL disruption. Taken together, these results indicate that the N207 glycosylation site is required for endothelial cell internalization via clathrin-mediated endocytosis and endosomal trafficking of NS1, which is necessary for the activation of enzymes such as cathepsin L that lead to EGL degradation and increased endothelial permeability (Fig 9A and 9B).