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      Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

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          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.

          Author summary

          Vascular leak is a hallmark of severe dengue disease. Recently, our group revealed a critical role for NS1 in induction of endothelial hyperpermeability and vascular leakage in an endothelial cell-intrinsic manner. However, the upstream pathway triggered by NS1, as well as the molecular determinants of NS1 required for this phenomenon, remain obscure. Gaining insight into this endothelial cell-intrinsic pathway is critical for understanding dengue pathogenesis, developing novel antiviral therapies, and developing NS1-based vaccine approaches that pose a minimal risk of antibody-dependent enhancement. Our current study expands our knowledge of this novel pathway not only by identifying the requirement of internalization of secreted NS1 via clathrin-mediated endocytosis, but also by pinpointing the NS1 molecular determinant (N207) required to trigger vascular leak. Further, our work identifies N207 as a residue conserved among multiple flaviviruses (Zika virus and West Nile virus, in addition to DENV), which is critical for NS1-mediated vascular leak in biologically relevant human endothelial cells modeling interstitial compartments in the lung or the blood-brain barrier. Thus, our study identifies endocytosis and a single amino acid (N207) of the NS1 viral toxin as critical for pan-flavivirus pathogenesis, representing a novel target for anti-flaviviral therapy and vaccine development.

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          Most cited references43

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          Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination.

          The four dengue virus serotypes (DENV1 to DENV4) are mosquito-borne flaviviruses that cause up to ~100 million cases of dengue annually worldwide. Severe disease is thought to result from immunopathogenic processes involving serotype cross-reactive antibodies and T cells that together induce vasoactive cytokines, causing vascular leakage that leads to shock. However, no viral proteins have been directly implicated in triggering endothelial permeability, which results in vascular leakage. DENV nonstructural protein 1 (NS1) is secreted and circulates in patients' blood during acute infection; high levels of NS1 are associated with severe disease. We show that inoculation of mice with DENV NS1 alone induces both vascular leakage and production of key inflammatory cytokines. Furthermore, simultaneous administration of NS1 with a sublethal dose of DENV2 results in a lethal vascular leak syndrome. We also demonstrate that NS1 from DENV1, DENV2, DENV3, and DENV4 triggers endothelial barrier dysfunction, causing increased permeability of human endothelial cell monolayers in vitro. These pathogenic effects of physiologically relevant amounts of NS1 in vivo and in vitro were blocked by NS1-immune polyclonal mouse serum or monoclonal antibodies to NS1, and immunization of mice with NS1 from DENV1 to DENV4 protected against lethal DENV2 challenge. These findings add an important and previously overlooked component to the causes of dengue vascular leak, identify a new potential target for dengue therapeutics, and support inclusion of NS1 in dengue vaccines.
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            Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization.

            Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.
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              Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition.

              Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: InvestigationRole: Methodology
                Role: Formal analysisRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                29 July 2019
                July 2019
                : 15
                : 7
                : e1007938
                Affiliations
                [001]Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, California, United States of America
                University of Pennsylvania School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-3050-4480
                http://orcid.org/0000-0003-1991-629X
                http://orcid.org/0000-0002-7225-2261
                http://orcid.org/0000-0001-8523-1104
                http://orcid.org/0000-0002-4289-1222
                http://orcid.org/0000-0002-4364-5031
                http://orcid.org/0000-0002-7238-4037
                Article
                PPATHOGENS-D-18-01905
                10.1371/journal.ppat.1007938
                6687192
                31356638
                d7ad5f96-6456-4959-bcae-74ad3a6347b3
                © 2019 Wang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 October 2018
                : 22 June 2019
                Page count
                Figures: 9, Tables: 0, Pages: 33
                Funding
                Funded by: NIAID/NIH
                Award ID: R01 AI24493
                Award Recipient :
                This work was supported by NIAID/NIH grant R01 AI24493 (E.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Endocytosis
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Secretory Pathway
                Endocytosis
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Epithelial Cells
                Endothelial Cells
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Endothelial Cells
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Endothelial Cells
                Biology and life sciences
                Organisms
                Viruses
                RNA viruses
                Flaviviruses
                West Nile virus
                Biology and life sciences
                Microbiology
                Medical microbiology
                Microbial pathogens
                Viral pathogens
                Flaviviruses
                West Nile virus
                Medicine and health sciences
                Pathology and laboratory medicine
                Pathogens
                Microbial pathogens
                Viral pathogens
                Flaviviruses
                West Nile virus
                Biology and life sciences
                Organisms
                Viruses
                Viral pathogens
                Flaviviruses
                West Nile virus
                Biology and Life Sciences
                Biochemistry
                Glycobiology
                Glycosylation
                Biology and Life Sciences
                Biochemistry
                Proteins
                Post-Translational Modification
                Glycosylation
                Physical Sciences
                Chemistry
                Chemical Compounds
                Salts
                Sulfates
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                Small interfering RNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Small interfering RNAs
                Biology and Life Sciences
                Cell Biology
                Cell Physiology
                Cell Binding
                Biology and Life Sciences
                Biochemistry
                Proteins
                Recombinant Proteins
                Custom metadata
                vor-update-to-uncorrected-proof
                2019-08-08
                All relevant data are within the manuscript and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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