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      Access to a main alphaherpesvirus receptor, located basolaterally in the respiratory epithelium, is masked by intercellular junctions

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          Abstract

          The respiratory epithelium of humans and animals is frequently exposed to alphaherpesviruses, originating from either external exposure or reactivation from latency. To date, the polarity of alphaherpesvirus infection in the respiratory epithelium and the role of respiratory epithelial integrity herein has not been studied. Equine herpesvirus type 1 (EHV1), a well-known member of the alphaherpesvirus family, was used to infect equine respiratory mucosal explants and primary equine respiratory epithelial cells (EREC), grown at the air-liquid interface. EHV1 binding to and infection of mucosal explants was greatly enhanced upon destruction of the respiratory epithelium integrity with EGTA or N-acetylcysteine. EHV1 preferentially bound to and entered EREC at basolateral cell surfaces. Restriction of infection via apical inoculation was overcome by disruption of intercellular junctions. Finally, basolateral but not apical EHV1 infection of EREC was dependent on cellular N-linked glycans. Overall, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host’s innate defence against primary alphaherpesvirus infections. In addition, by targeting a basolaterally located receptor in the respiratory epithelium, alphaherpesviruses have generated a strategy to efficiently escape from host defence mechanisms during reactivation from latency.

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          Most cited references 81

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          Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical- basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein

          Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.
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            Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology.

            Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.
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              Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

              The epithelial cadherin (E-cadherin)–catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell’s apical–basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling.
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                Author and article information

                Contributors
                hans.nauwynck@ugent.be
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                30 November 2017
                30 November 2017
                2017
                : 7
                Affiliations
                [1 ]ISNI 0000 0001 2069 7798, GRID grid.5342.0, Department of Virology, Parasitology and Immunology, , Faculty of Veterinary Medicine, Ghent University, ; Salisburylaan 133, 9820 Merelbeke, Belgium
                [2 ]ISNI 0000 0001 2097 5006, GRID grid.16750.35, Department of Molecular Biology and Princeton Neuroscience Institute, , Princeton University, ; 119 Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544 USA
                [3 ]ISNI 0000 0001 2150 1785, GRID grid.17088.36, Department of Pathobiology and Diagnostic Investigation, , College of Veterinary Medicine, Michigan State University, ; 784 Wilson Road, East Lansing, Michigan 48824 USA
                [4 ]ISNI 0000 0001 2069 7798, GRID grid.5342.0, Department of Morphology, , Faculty of Veterinary Medicine, Ghent University, ; Salisburylaan 133, 9820 Merelbeke, Belgium
                Article
                16804
                10.1038/s41598-017-16804-5
                5709510
                29192251
                © The Author(s) 2017

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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