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Patricia G. Spear - One of the best experts on this subject based on the ideXlab platform.
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infection of neurons and encephalitis after intracranial inoculation of herpes simplex virus requires the entry receptor Nectin 1
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Sarah J. Kopp, Ghazal Banisadr, Kelly E Glajch, Ulrike E Maurer, Kay Grunewald, Richard J Miller, Pavel Osten, Patricia G. SpearAbstract:Multiple entry receptors can mediate infection of cells by herpes simplex virus (HSV), permitting alternative pathways for infection and disease. We investigated the roles of two known entry receptors, herpesvirus entry mediator (HVEM) and Nectin-1, in infection of neurons in the CNS and the development of encephalitis. Wild-type, HVEM KO, Nectin-1 KO, and HVEM/Nectin-1 double KO mice were inoculated with HSV into the hippocampus. The mice were examined for development of encephalitis or were killed at various times after inoculation for immunohistological analyses of brain slices. Nectin-1 KO mice showed no signs of disease after intracranial inoculation, and no HSV antigens were detectable in the brain parenchyma. However, HSV antigens were detected in non-parenchymal cells lining the ventricles. In the double KO mice, there was also no disease and no detectable expression of viral antigens even in non-parenchymal cells, indicating that infection of these cells in the Nectin-1 KO mice was dependent on the expression of HVEM. Wild-type and HVEM KO mice rapidly developed encephalitis, and the patterns of HSV replication in the brain were indistinguishable. Thus, expression of Nectin-1 is necessary for HSV infection via the intracranial route and for encephalitis; HVEM is largely irrelevant. These results contrast with recent findings that (i) either HVEM or Nectin-1 can permit HSV infection of the vaginal epithelium in mice and (ii) Nectin-1 is not the sole receptor capable of enabling spread of HSV infection from the vaginal epithelium to the PNS and CNS.
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Alternative Entry Receptors for Herpes Simplex Virus and Their Roles in Disease
Cell host & microbe, 2007Co-Authors: Joann M. Taylor, Jun Miyoshi, Yoshimi Takai, Miri Yoon, Anna Zago, Erick Lin, Nanette Susmarski, Carl F. Ware, Klaus Pfeffer, Patricia G. SpearAbstract:Either herpesvirus entry mediator (HVEM, TNFRSF14) or Nectin-1 (PVRL1) is sufficient for herpes simplex virus (HSV) infection of cultured cells. The contribution of individual receptors to infection in vivo and to disease is less clear. To assess this, Tnfrsf14(-/-) and/or Pvrl1(-/-) mice were challenged intravaginally with HSV-2. Infection of the vaginal epithelium occurred in the absence of either HVEM or Nectin-1 but was virtually undetectable when both receptors were absent, indicating that either HVEM or Nectin-1 was necessary. Absence of Nectin-1 (but not HVEM) reduced efficiency of infection of the vaginal epithelium and viral spread to the nervous system, attenuating neurological disease and preventing external lesion development. While Nectin-1 proved not to be essential for infection of the nervous system, it is required for the full manifestations of disease. This study illustrates the value of mutant mice for understanding receptor contributions to disease caused by a human virus.
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Deletion of the Second Immunoglobulin-Like Domain of Nectin-1 Alters Its Intracellular Processing and Localization and Ability To Mediate Entry of Herpes Simplex Virus
Journal of virology, 2005Co-Authors: Frank Struyf, Aileen E. Plate, Patricia G. SpearAbstract:Nectin-1 is an immunoglobulin (Ig)-like entry receptor for herpes simplex virus (HSV). Like other Nectins, Nectin-1 forms dimers and mediates cell adhesion through interactions with other Nectins. We constructed a second-domain deletion mutant of Nectin-1 (Nectin-1-Δ2) to examine the role of the second Ig-like domain in HSV entry. Nectin-1-Δ2 exhibited a severely reduced ability to mediate HSV entry and accumulated in the endoplasmic reticulum but retained the ability to interact with its HSV ligand, gD. The failure of Nectin-1-Δ2 to mediate HSV entry probably resulted from its failure to be transported to a membrane targeted by HSV for viral entry.
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Effects of linker-insertion mutations in herpes simplex virus 1 gD on glycoprotein-induced fusion with cells expressing HVEM or Nectin-1.
Virology, 2004Co-Authors: Cheryl R. Jogger, Rebecca I. Montgomery, Patricia G. SpearAbstract:Several cell surface molecules, including HVEM and Nectin-1, can serve as entry receptors for herpes simplex virus (HSV) and as receptors for virus-induced or viral glycoprotein-induced cell fusion. The viral ligand for these receptors is the HSV envelope glycoprotein gD. A set of linker-insertion and deletion mutants of HSV type 1 (HSV-1) gD was analyzed for effects of the mutations on binding of gD to HVEM and Nectin-1, on viral glycoprotein-induced cell fusion with target cells expressing HVEM or Nectin-1 and on complementation of infectivity of a gD-null HSV-1 viral mutant. Insertions after amino acid 151 or 225 or deletion of amino acids 234–244 disrupted (i) binding of the mutant forms of gD to both receptors and (ii) functional interactions (cell fusion and complementation) with both receptors, but were without effect on cell surface expression. Insertions in the N-terminal domain of gD (after amino acid 12, 34 or 43) disrupted binding to HVEM and functional activities with HVEM, as expected from a previously reported X-ray structure of a gD–HVEM complex, but were without effect in the case of Nectin-1. These and other results indicate that the mutations disruptive of interactions with both receptors probably affect conformations of contact sites that are different for each receptor.
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Mutations in the N termini of herpes simplex virus type 1 and 2 gDs alter functional interactions with the entry/fusion receptors HVEM, Nectin-2, and 3-O-sulfated heparan sulfate but not with Nectin-1.
Journal of virology, 2003Co-Authors: Miri Yoon, D. Shukla, Anna Zago, Patricia G. SpearAbstract:Multiple cell surface molecules (herpesvirus entry mediator [HVEM], Nectin-1, Nectin-2, and 3-O-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except Nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for Nectin-1. Each of these three substitutions in HSV-1 gD enhanced fusion with cells expressing human Nectin-2 (ordinarily low for wild-type HSV-1 gD), but the same substitutions in HSV-2 gD were without effect on the already high level of cell fusion observed with the wild-type protein. The Q27P or Q27R substitution in either HSV-1 and HSV-2 gD, but not the L25P substitution, significantly reduced cell fusion and binding activity for both human and mouse HVEM. Each of the three substitutions in HSV-1 gD, as well as the deletions mentioned above, reduced fusion with cells bearing 3-O-sulfated heparan sulfate. Thus, the N terminus of HSV-1 or HSV-2 gD is not necessary for functional interactions with Nectin-1 but is necessary for all of the other receptors tested here. The sequence of the N terminus determines whether Nectin-2 or 3-O-sulfated heparan sulfate, as well as HVEM, can serve as entry/fusion receptors.
Claude Krummenacher - One of the best experts on this subject based on the ideXlab platform.
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Effect on Nectin-1 on susceptibility to NK cell cytotoxicity.
2019Co-Authors: Veronica M. Holmes, Arjun K. Bhargava, Carlos Maluquer De Motes, Paige T. Richards, Jessenia Roldan, Jordan S. Orange, Claude KrummenacherAbstract:Wild type K562 cells or K562-N1G cells, which expresses GFP-Nectin-1 were labeled with 51Cr and mixed with effector NK-92 cells at the indicated ratio and incubated for 4 h. Release of 51Cr is measured and % specific release is compared to K562 and K562-N1G in the absence of NK cells (0% lysis) or lysed with detergent (100% lysis). (A) Data from a representative experiment shows average of triplicate measurements ± one standard deviation. (B) Combined data from four independent experiments show the relative killing of K562-NG1 compared to wild type K562 (normalized to 100%) at different effector to target ratios. Averages values ± one standard deviation are shown. Significance of differences between cell types at each effector to target ratio was determined by one way ANOVA (**P
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Affinity and kinetics constants of CD96(517t) ectodomain to Nectin-1(346t) ectodomain and Nectin-1(143t) V-domain determined by SPR.
2019Co-Authors: Veronica M. Holmes, Arjun K. Bhargava, Carlos Maluquer De Motes, Paige T. Richards, Jessenia Roldan, Jordan S. Orange, Claude KrummenacherAbstract:Affinity and kinetics constants of CD96(517t) ectodomain to Nectin-1(346t) ectodomain and Nectin-1(143t) V-domain determined by SPR.
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Expression of GFP-Nectin-1 in human K562 cells.
2019Co-Authors: Veronica M. Holmes, Arjun K. Bhargava, Carlos Maluquer De Motes, Paige T. Richards, Jessenia Roldan, Jordan S. Orange, Claude KrummenacherAbstract:(A) K562 cells transiently transfected with plasmid pCK495 express Nectin-1 with GFP tagged at the N-terminus. GFP-Nectin-1 is expressed at the cell surface and accumulates at cell-cell contacts. Images of GFP fluorescence, magnification 40x. (B) Flow cytometry analysis of K562 cells stably transfected to express GFP-Nectin-1. Cells were stained with PE-tagged anti-Nectin-1 antibody CK41. Left histogram: K562 cells stained with CK41-PE (red line) are compared to control (gray shade). Right panel: cells from clone K562-N1G #11 expressing GFP-Nectin-1 were stained with CK41-PE (red lines vs control shaded in gray). (C) Wild type K562 cells and K562-N1G cells were exposed to lacZ reporter virus HSV-1 KOStk12 at the indicated MOI. Activity of the virus-encoded beta-galactosidase was recorded as the change of OD570nm over time to reflect virus entry. Values from at least two independent experiments were normalized to the highest value (K562-N1G cells at MOI = 40) set at 100% and averaged. Error bars indicate standard deviations across independent experiments.
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Interaction between human CD96 and Nectin-1 V-domain by ELISA.
2019Co-Authors: Veronica M. Holmes, Arjun K. Bhargava, Carlos Maluquer De Motes, Paige T. Richards, Jessenia Roldan, Jordan S. Orange, Claude KrummenacherAbstract:Plates were coated with Nectin-1(143t)-MBP at 10 μg/ml, or mock treated (milk protein control). Increasing concentrations of hCD96t were added and bound hCD96t was detected using an anti-tetraHis tag Mab (Qiagen), followed by secondary antibody and substrate. Absorbance was read at 405 nm. Average values ± one standard deviation of four independent experiments are shown.
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Interaction between Nectin-1 and the human natural killer cell receptor CD96
2019Co-Authors: Veronica M. Holmes, Arjun K. Bhargava, Carlos Maluquer De Motes, Paige T. Richards, Jessenia Roldan, Jordan S. Orange, Claude KrummenacherAbstract:Regulation of Natural Killer (NK) cell activity is achieved by the integration of both activating and inhibitory signals acquired at the immunological synapse with potential target cells. NK cells express paired receptors from the immunoglobulin family which share common ligands from the Nectin family of adhesion molecules. The activating receptor CD226 (DNAM-1) binds to Nectin-2 and CD155, which are also recognized by the inhibitory receptor TIGIT. The third receptor in this family is CD96, which is less well characterized and may have different functions in human and mouse models. Human CD96 interacts with CD155 and ligation of this receptor activates NK cells, while in mice the presence of CD96 correlates with decreased NK cell activation. Mouse CD96 also binds Nectin-1, but the effect of this interaction has not yet been determined. Here we show that human Nectin-1 directly interacts with CD96 in vitro. The binding site for CD96 is located on the Nectin-1 V-domain, which comprises a canonical interface that is shared by Nectins to promote cell adhesion. The affinity of Nectin-1 for CD96 is lower than for other Nectins such as Nectin-3 and Nectin-1 itself. However, the affinity of Nectin-1 for CD96 is similar to its affinity for herpes simplex virus glycoprotein D (HSV gD), which binds the Nectin-1 V-domain during virus entry. The affinity of human CD96 for Nectin-1 is lower than for its known activating ligand CD155. We also found that human erythroleukemia K562 cells, which are commonly used as susceptible targets to assess NK cell cytotoxicity did not express Nectin-1 on their surface and were resistant to HSV infection. When expressed in K562 cells, Nectin-1-GFP accumulated at cell contacts and allowed HSV entry. Furthermore, overexpression of Nectin-1-GFP led to an increased susceptibility of K562 cells to NK-92 cell cytotoxicity.
Roselyn J Eisenberg - One of the best experts on this subject based on the ideXlab platform.
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Induction of conformational changes at the N-terminus of herpes simplex virus glycoprotein D upon binding to HVEM and Nectin-1.
Virology, 2013Co-Authors: Eric Lazear, Roselyn J Eisenberg, Gary H Cohen, Andrea Carfi, J. Charles Whitbeck, Yi Zuo, Claude KrummenacherAbstract:Herpes simplex virus entry is initiated by glycoprotein D (gD) binding to a cellular receptor, such as HVEM or Nectin-1. gD is activated by receptor-induced displacement of the C-terminus from the core of the glycoprotein. Binding of HVEM requires the formation of an N-terminal hairpin loop of gD; once formed this loop masks the Nectin-1 binding site on the core of gD. We found that HVEM and Nectin-1 exhibit non-reciprocal competition for binding to gD. The N-terminus of gD does not spontaneously form a stable hairpin in the absence of receptor and HVEM does not appear to rely on a pre-existing hairpin for binding to gD(3C-38C) mutants. However, HVEM function is affected by mutations that impair optimal hairpin formation. Furthermore, Nectin-1 induces a new conformation of the N-terminus of gD. We conclude that the conformation of the N-terminus of gD is actively modified by the direct action of both receptors.
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structure of herpes simplex virus glycoprotein d bound to the human receptor Nectin 1
PLOS Pathogens, 2011Co-Authors: Paolo Di Giovine, Claude Krummenacher, Roselyn J Eisenberg, Gary H Cohen, Arjun K. Bhargava, Ethan C Settembre, Micah A Luftig, Huan Lou, Andrea CarfiAbstract:Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to Nectin-1 determined by x-ray crystallography to 4.0 A resolution. The structure reveals that the Nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of Nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop conNecting β-strands F and G of Nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents Nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal Nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.
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The herpes simplex virus receptor Nectin-1 is down-regulated after trans-interaction with glycoprotein D
Virology, 2008Co-Authors: Katie M. Stiles, Gary H Cohen, Roselyn J Eisenberg, Richard S. B. Milne, Claude KrummenacherAbstract:During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as Nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with Nectin-1 expressing cells to investigate the effects of gD on Nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of Nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, Nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that Nectin-1 down-regulation corresponds to the ability of gD to bind Nectin-1 and is achieved by internalization and low-pH-dependent degradation of Nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing Nectin-1. These data suggest that the determinants of gD-mediated internalization of Nectin-1 may direct HSV to an endocytic pathway during entry.
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Spatiotemporal changes of the herpes simplex virus entry receptor Nectin-1 in murine brain during postnatal development
Journal of NeuroVirology, 2006Co-Authors: Szatmár Horváth, Emese Prandovszky, Claude Krummenacher, Roselyn J Eisenberg, Gary H Cohen, Zoltan Janka, József ToldiAbstract:Herpes simplex virus (HSV) is known to replicate within the limbic system and to alter behavior in both humans and experimental animals. However, the reason why the virus selectively damages this anatomical, developmental, and functional neural unit remains a mystery. Nor is it known why herpes simplex encephalitis fails to respect these neuroanatomical boundaries in newborns. In the present study, the authors determined the spatiotemporal changes in the distribution of the major neural entry receptor for HSV (Nectin-1) in postnatal mouse and rat brains. Discrete Nectin-1 immunopositivity was observed in regions susceptible to HSV infection in specific developmental phases of central nervous system. The authors also describe Nectin-1-related pathways controlling neuronal cell migration/brain morphogenesis, the disruption of which might lead to the emergence of mental disorders with a rapid cognitive decline.
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Potential Nectin-1 binding site on herpes simplex virus glycoprotein d.
Journal of virology, 2005Co-Authors: Sarah A. Connolly, Gary H Cohen, Andrea Carfi, J. Charles Whitbeck, Daniel J. Landsburg, Yi Zuo, Don C. Wiley, Roselyn J EisenbergAbstract:Four glycoproteins (gD, gB, gH, and gL) are essential for herpes simplex virus (HSV) entry into cells. An early step of fusion requires gD to bind one of several receptors, such as Nectin-1 or herpesvirus entry mediator (HVEM). We hypothesize that a conformational change in gD occurs upon receptor binding that triggers the other glycoproteins to mediate fusion. Comparison of the crystal structures of gD alone and gD bound to HVEM reveals that upon HVEM binding, the gD N terminus transitions from a flexible stretch of residues to a hairpin loop. To address the contribution of this transition to the ability of gD to trigger fusion, we attempted to “lock” the gD N terminus into a looped conformation by engineering a disulfide bond at its N and C termini. The resulting mutant (gD-A3C/Y38C) failed to trigger fusion in the absence of receptor, suggesting that formation of the loop is not the sole fusion trigger. Unexpectedly, although gD-A3C/Y38C bound HVEM, it failed to bind Nectin-1. This was due to the key role played by Y38 in interacting with Nectin-1. Since tyrosines are often “hot spot” residues at the center of protein-protein interfaces, we mutated residues that surround Y38 on the same face of gD and tested their binding and functional properties. Our results suggest that this region of gD is important for Nectin-1 interaction and is distinct from but partially overlaps the site of HVEM binding. Unique gD mutants with altered receptor usage generated in this study may help dissect the roles played by various HSV receptors during infection.
Tibor Valyi-nagy - One of the best experts on this subject based on the ideXlab platform.
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Nectin-1-specific entry of herpes simplex virus 1 is sufficient for infection of the cornea and viral spread to the trigeminal ganglia
Molecular vision, 2012Co-Authors: Navika D. Shukla, Vaibhav Tiwari, Tibor Valyi-nagyAbstract:Purpose Primary and recurrent infections of the cornea by herpes simplex virus 1 (HSV-1) are important causes of eye disease. Three unrelated classes of glycoprotein D receptors for HSV-1 entry into cells have been identified. This study was undertaken to uncover the relative significance of Nectin-1 as an entry receptor in corneal infection and HSV-1 spread to the trigeminal ganglia (TG), a site important for HSV-1 latency and recurrent corneal infection. Methods To assess the significance of Nectin-1, a member of the immunoglobulin superfamily, in primary HSV-1 infection and spread to the TG, we used a murine model of corneal infection and a HSV-1 mutant, KOS(Rid1), which can only use Nectin-1 for entry. Immunohistochemistry, real-time PCR, and plaque assays using HSV-1 infected tissues were performed. Results We demonstrated that receptor usage by HSV-1 limited to Nectin-1 does not significantly change the spread of HSV-1 in the corneal epithelium during primary infection. We also found that Nectin-1-specific entry does not affect the capacity of the virus to spread to the TG from the cornea. Conclusions Our findings suggest that Nectin-1 alone is sufficient for HSV-1 entry into the cornea and spread to the TG.
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Increased axonal expression of Nectin-1 in multiple sclerosis plaques
Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology, 2012Co-Authors: Karla J. Castellanos, Szatmár Horváth, D. Shukla, K. V. Slavin, Eva Gagyi, Bernadett Kormos, Klara Valyi-nagy, Andras Voros, Tibor Valyi-nagyAbstract:Nectin-1 is a cell adhesion molecule that plays a role in interneuronal synapse formation, in axonal guidance during development and possibly in neuron-glia interactions. To better understand axonal changes in MS, Nectin-1 expression was determined by immunohistochemistry in normal adult human cerebral white matter (n = 4) and in six MS plaques (three active and three inactive). The intensity of axonal Nectin-1 expression was scored on a scale of 0 to 4+. In normal adult cerebral white matter, axons showed weak Nectin-1 expression with a score of 1.25 ± 0.50. Axonal Nectin-1 expression was significantly stronger within both active (score = 3.33 ± 0.289, p = 0.001) and inactive (score = 2.16 ± 0.29, p = 0.038) MS plaques than in normal white matter. Axons in white matter adjacent to MS plaques showed Nectin-1 expression (score = 1.5 ± 0.50) that was not statistically different from normal controls (p = 0.542). These findings raise the possibility that increased expression of Nectin-1 in MS lesions plays a role in the pathogenesis of MS through participation in axonal responses to injury and mediation of altered neuron-glia interactions relevant to myelination.
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Role for Nectin-1 in herpes simplex virus 1 entry and spread in human retinal pigment epithelial cells.
The FEBS journal, 2008Co-Authors: Vaibhav Tiwari, Tibor Valyi-nagy, Mária Kovács, Shripaad Y. Shukla, D. ShuklaAbstract:Herpes simplex virus 1 (HSV-1) demonstrates a unique ability to infect a variety of host cell types. Retinal pigment epithelial (RPE) cells form the outermost layer of the retina and provide a potential target for viral invasion and permanent vision impairment. Here we examine the initial cellular and molecular mechanisms that facilitate HSV-1 invasion of human RPE cells. High-resolution confocal microscopy demonstrated initial interaction of green fluorescent protein (GFP)-tagged virions with filopodia-like structures present on cell surfaces. Unidirectional movement of the virions on filopodia to the cell body was detected by live cell imaging of RPE cells, which demonstrated susceptibility to pH-dependent HSV-1 entry and replication. Use of RT-PCR indicated expression of Nectin-1, herpes virus entry mediator (HVEM) and 3-O-sulfotransferase-3 (as a surrogate marker for 3-O-sulfated heparan sulfate). HVEM and Nectin-1 expression was subsequently verified by flow cytometry. Nectin-1 expression in murine retinal tissue was also demonstrated by immunohistochemistry. Antibodies against Nectin-1, but not HVEM, were able to block HSV-1 infection. Similar blocking effects were seen with a small interfering RNA construct specifically directed against Nectin-1, which also blocked RPE cell fusion with HSV-1 glycoprotein-expressing Chinese hamster ovary (CHO-K1) cells. Anti-Nectin-1 antibodies and F-actin depolymerizers were also successful in blocking the cytoskeletal changes that occur upon HSV-1 entry into cells. Our findings shed new light on the cellular and molecular mechanisms that help the virus to enter the cells of the inner eye.
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HVEM and Nectin-1 Are the Major Mediators of Herpes Simplex Virus 1 (HSV-1) Entry into Human Conjunctival Epithelium
Investigative ophthalmology & visual science, 2008Co-Authors: Jihan Akhtar, Tibor Valyi-nagy, Vaibhav Tiwari, Mária Kovács, S. Krisztian Kovacs, Aarti Jani, D. ShuklaAbstract:Herpes simplex virus (HSV)-1, a member of the alphaherpesvirus subfamily, can infect a variety of ocular cell types and is the most common cause of infectious blindness in the developed world.1,2 HSV-1 infection of the eye results in various diseases such as stromal keratitis, epithelial keratitis, and conjunctivitis.2–4 HSV-1 is the most frequently isolated virus in ocular infections, resulting in 20,000 primary and 28,000 reactivated ocular infections per year in the United States. In patients with conjunctivitis, HSV-1 is the second most common laboratory isolated virus and is responsible for approximately 21% of cases of acute viral conjunctivitis.1,2 The conjunctiva is responsible for protecting the eye and maintaining the moisture within it, and its infection can lead to serious complications.3,4 Conjunctival epithelium is also directly continuous with corneal epithelium, thus facilitating the spread of virus.5 This may ultimately lead to blindness through corneal scarring caused by recurrent infections from latent HSV-1.2,6 Detection and treatment of conjunctival HSV-1 infection is important because the frequency of recurrence has been shown to correlate highly with conjunctival severity compared with corneal severity. Reactivation of HSV-1 more often involves the conjunctiva, whereas reactivation infection of the cornea is rare.2 HSV-1 is an enveloped DNA virus that requires at least five envelope glycoproteins to enter host cells.7 Viral glycoproteins gB and gC first attach to heparan sulfate proteoglycans on the cell surface.8 After this activity, a conformational change brings glycoprotein D (gD) in the proximity of a gD receptor on the host cell.7 A collaborative effort among gD, the gD receptor, gB, gH, gL, and possibly an additional interaction of gH with other cell surface molecules, then leads to fusion of a cell membrane and viral envelope.7,9 Fusion with the plasma membrane is thought to be pH independent. However, depending on the cell type, HSV-1 can enter in a pH-dependent fashion and can fuse with the membrane of an intracellular vesicle.10 There are three distinct classes of known HSV-1 entry receptors.7 Among these, herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor receptor family.11 Nectin-1 (HveC) and Nectin-2 (HveB) belong to the immunoglobulin superfamily. Nectin-2 is unique because it mediates the entry of some laboratory generated mutants but not wild-type strains of HSV-1.7 Lastly, specific sites on heparan sulfate, when modified by certain isoforms of 3-O-sulfotransferase (3-OST) such as 3-OST-3, 3-OST-5, and 3-OST-6, also work as entry receptors for HSV-1.12–14 The use of entry receptors varies with cell type. HVEM is the main entry receptor for human T lymphocytes and trabecular meshwork cells.11,15 Nectin-1 is thought to be important for the infection of cells of epithelial and neuronal origin.16,17 3-O–sulfated heparan sulfate (3-OS HS) is the major mediator of entry into corneal fibroblasts.18 Although conjunctiva is a target of HSV-1 infection, the identity of functional HSV-1 entry receptors in the cells of the conjunctiva remains unknown. Similarly, the pH dependence of entry remains undetermined. In the present study, we sought to examine the molecular and cellular aspects of initial interaction of HSV-1 virions with human conjunctival epithelial (HCjE) cells leading to HSV-1 entry and replication. This study demonstrates the pH-dependent nature of HSV-1 entry, implicating endocytosis in viral uptake. It also demonstrates that the expression of Nectin-1 and HVEM predominantly determines HSV-1 entry into HCjE cells. This is the first report of a cell type in which both Nectin-1 and HVEM are found critical for entry.
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Nectin-1 (HveC) is expressed at high levels in neural subtypes that regulate radial migration of cortical and cerebellar neurons of the developing human and murine brain
Journal of neurovirology, 2008Co-Authors: Emese Prandovszky, Szatmár Horváth, Zoltan Janka, József Toldi, D. Shukla, Levente Gellért, S. Krisztian Kovacs, Tibor Valyi-nagyAbstract:Herpes simplex viruses (HSV) produce age-dependent encephalitis characterized by more severe involvement of the cerebral cortex in younger hosts. To elucidate the potential role of the major neural entry receptor of HSV, Nectin-1, in age-dependent susceptibility of cortical neurons to viral encephalitis, the authors examined the anatomical distribution of the receptor protein in the developing human and mouse cerebral cortex, hippocampus, and cerebellum by immunohistochemistry. Nectin-1 is expressed at high levels in guiding cells (radial glial cells and Cajal-Retzius cells) that regulate radial migration of neurons in cortical lamination, at lower levels in migrating neurons, and at variable levels in the transient ventricular and marginal zones of the cerebral cortical wall. These results may have implications regarding the selective spatiotemporal tropism of HSV to specific neuronal populations, and for the better understanding of neurodevelopmental defects caused by fetal HSV infections.
D. Shukla - One of the best experts on this subject based on the ideXlab platform.
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Increased axonal expression of Nectin-1 in multiple sclerosis plaques
Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology, 2012Co-Authors: Karla J. Castellanos, Szatmár Horváth, D. Shukla, K. V. Slavin, Eva Gagyi, Bernadett Kormos, Klara Valyi-nagy, Andras Voros, Tibor Valyi-nagyAbstract:Nectin-1 is a cell adhesion molecule that plays a role in interneuronal synapse formation, in axonal guidance during development and possibly in neuron-glia interactions. To better understand axonal changes in MS, Nectin-1 expression was determined by immunohistochemistry in normal adult human cerebral white matter (n = 4) and in six MS plaques (three active and three inactive). The intensity of axonal Nectin-1 expression was scored on a scale of 0 to 4+. In normal adult cerebral white matter, axons showed weak Nectin-1 expression with a score of 1.25 ± 0.50. Axonal Nectin-1 expression was significantly stronger within both active (score = 3.33 ± 0.289, p = 0.001) and inactive (score = 2.16 ± 0.29, p = 0.038) MS plaques than in normal white matter. Axons in white matter adjacent to MS plaques showed Nectin-1 expression (score = 1.5 ± 0.50) that was not statistically different from normal controls (p = 0.542). These findings raise the possibility that increased expression of Nectin-1 in MS lesions plays a role in the pathogenesis of MS through participation in axonal responses to injury and mediation of altered neuron-glia interactions relevant to myelination.
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Increased axonal expression of Nectin-1 in multiple sclerosis
2012Co-Authors: D. Shukla, S. Horvath, K. V. SlavinAbstract:Nectin-1 is a cell adhesion molecule that plays a role in interneuronal synapse formation, in axonal guid- ance during development and possibly in neuron-glia interactions. To better understand axonal changes in MS, Nectin-1 expression was determined by immunohisto- chemistry in normal adult human cerebral white matter (n = 4) and in six MS plaques (three active and three inactive). The intensity of axonal Nectin-1 expression was scored on a scale of 0 to 4?. In normal adult cerebral white matter, axons showed weak Nectin-1 expression with a score of 1.25 ± 0.50. Axonal Nectin-1 expression was significantly stronger within both active (score = 3.33 ± 0.289, p = 0.001) and inactive (score = 2.16 ± 0.29, p = 0.038) MS plaques than in normal white matter. Axons in white matter adjacent to MS plaques showed Nectin-1 expression (score = 1.5 ± 0.50) that was not statistically different from normal controls (p = 0.542). These findings raise the possibility that increased expres- sion of Nectin-1 in MS lesions plays a role in the patho- genesis of MS through participation in axonal responses to injury and mediation of altered neuron-glia interactions relevant to myelination.
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Role of Nectin-1, HVEM, and PILR-α in HSV-2 Entry into Human Retinal Pigment Epithelial Cells
Investigative ophthalmology & visual science, 2009Co-Authors: Shripaad Y. Shukla, Yogesh K. Singh, D. ShuklaAbstract:Purpose Herpes simplex virus-type 2 (HSV-2) can cause acute retinal necrosis (ARN), which can lead to exudative and rhegmatogenous retinal detachment, yet little is known about the cellular and molecular mechanisms of HSV-2 entry into retinal pigment epithelial (RPE) cells. The goal of this study was to establish the identity of the critical receptors used by the virus for infection. Methods A reporter HSV-2 virus, which expresses beta-galactosidase, was used to quantify entry into RPE cells, and viral replication was ascertained using a plaque assay. Flow cytometry and immunocytochemistry were used to determine cellular expression of entry receptors. Localization of these receptors to the apical or basal surface of RPE cells was determined with immunocytochemistry. The necessity of these receptors, individually and in combination, for viral entry was established using receptor-specific antibodies and siRNAs. Results RPE cells are highly susceptible to HSV-2 entry and replication. Several assays demonstrated the expression of the entry receptors Nectin-1, HVEM, and PILR-alpha and their localization primarily to the apical surfaces of RPE cells. Receptor-specific antibodies and siRNA knockdown of receptors significantly reduced viral entry and implicated Nectin-1 as an important receptor, with HVEM and PILR-alpha potentially also contributing to entry. Conclusions HSV-2 is capable of developing a productive infection in RPE cells by using Nectin-1 as an important entry receptor. To lesser degrees, HVEM and PILR-alpha may also contribute to HSV-2 entry into RPE cells.
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Role for Nectin-1 in herpes simplex virus 1 entry and spread in human retinal pigment epithelial cells.
The FEBS journal, 2008Co-Authors: Vaibhav Tiwari, Tibor Valyi-nagy, Mária Kovács, Shripaad Y. Shukla, D. ShuklaAbstract:Herpes simplex virus 1 (HSV-1) demonstrates a unique ability to infect a variety of host cell types. Retinal pigment epithelial (RPE) cells form the outermost layer of the retina and provide a potential target for viral invasion and permanent vision impairment. Here we examine the initial cellular and molecular mechanisms that facilitate HSV-1 invasion of human RPE cells. High-resolution confocal microscopy demonstrated initial interaction of green fluorescent protein (GFP)-tagged virions with filopodia-like structures present on cell surfaces. Unidirectional movement of the virions on filopodia to the cell body was detected by live cell imaging of RPE cells, which demonstrated susceptibility to pH-dependent HSV-1 entry and replication. Use of RT-PCR indicated expression of Nectin-1, herpes virus entry mediator (HVEM) and 3-O-sulfotransferase-3 (as a surrogate marker for 3-O-sulfated heparan sulfate). HVEM and Nectin-1 expression was subsequently verified by flow cytometry. Nectin-1 expression in murine retinal tissue was also demonstrated by immunohistochemistry. Antibodies against Nectin-1, but not HVEM, were able to block HSV-1 infection. Similar blocking effects were seen with a small interfering RNA construct specifically directed against Nectin-1, which also blocked RPE cell fusion with HSV-1 glycoprotein-expressing Chinese hamster ovary (CHO-K1) cells. Anti-Nectin-1 antibodies and F-actin depolymerizers were also successful in blocking the cytoskeletal changes that occur upon HSV-1 entry into cells. Our findings shed new light on the cellular and molecular mechanisms that help the virus to enter the cells of the inner eye.
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HVEM and Nectin-1 Are the Major Mediators of Herpes Simplex Virus 1 (HSV-1) Entry into Human Conjunctival Epithelium
Investigative ophthalmology & visual science, 2008Co-Authors: Jihan Akhtar, Tibor Valyi-nagy, Vaibhav Tiwari, Mária Kovács, S. Krisztian Kovacs, Aarti Jani, D. ShuklaAbstract:Herpes simplex virus (HSV)-1, a member of the alphaherpesvirus subfamily, can infect a variety of ocular cell types and is the most common cause of infectious blindness in the developed world.1,2 HSV-1 infection of the eye results in various diseases such as stromal keratitis, epithelial keratitis, and conjunctivitis.2–4 HSV-1 is the most frequently isolated virus in ocular infections, resulting in 20,000 primary and 28,000 reactivated ocular infections per year in the United States. In patients with conjunctivitis, HSV-1 is the second most common laboratory isolated virus and is responsible for approximately 21% of cases of acute viral conjunctivitis.1,2 The conjunctiva is responsible for protecting the eye and maintaining the moisture within it, and its infection can lead to serious complications.3,4 Conjunctival epithelium is also directly continuous with corneal epithelium, thus facilitating the spread of virus.5 This may ultimately lead to blindness through corneal scarring caused by recurrent infections from latent HSV-1.2,6 Detection and treatment of conjunctival HSV-1 infection is important because the frequency of recurrence has been shown to correlate highly with conjunctival severity compared with corneal severity. Reactivation of HSV-1 more often involves the conjunctiva, whereas reactivation infection of the cornea is rare.2 HSV-1 is an enveloped DNA virus that requires at least five envelope glycoproteins to enter host cells.7 Viral glycoproteins gB and gC first attach to heparan sulfate proteoglycans on the cell surface.8 After this activity, a conformational change brings glycoprotein D (gD) in the proximity of a gD receptor on the host cell.7 A collaborative effort among gD, the gD receptor, gB, gH, gL, and possibly an additional interaction of gH with other cell surface molecules, then leads to fusion of a cell membrane and viral envelope.7,9 Fusion with the plasma membrane is thought to be pH independent. However, depending on the cell type, HSV-1 can enter in a pH-dependent fashion and can fuse with the membrane of an intracellular vesicle.10 There are three distinct classes of known HSV-1 entry receptors.7 Among these, herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor receptor family.11 Nectin-1 (HveC) and Nectin-2 (HveB) belong to the immunoglobulin superfamily. Nectin-2 is unique because it mediates the entry of some laboratory generated mutants but not wild-type strains of HSV-1.7 Lastly, specific sites on heparan sulfate, when modified by certain isoforms of 3-O-sulfotransferase (3-OST) such as 3-OST-3, 3-OST-5, and 3-OST-6, also work as entry receptors for HSV-1.12–14 The use of entry receptors varies with cell type. HVEM is the main entry receptor for human T lymphocytes and trabecular meshwork cells.11,15 Nectin-1 is thought to be important for the infection of cells of epithelial and neuronal origin.16,17 3-O–sulfated heparan sulfate (3-OS HS) is the major mediator of entry into corneal fibroblasts.18 Although conjunctiva is a target of HSV-1 infection, the identity of functional HSV-1 entry receptors in the cells of the conjunctiva remains unknown. Similarly, the pH dependence of entry remains undetermined. In the present study, we sought to examine the molecular and cellular aspects of initial interaction of HSV-1 virions with human conjunctival epithelial (HCjE) cells leading to HSV-1 entry and replication. This study demonstrates the pH-dependent nature of HSV-1 entry, implicating endocytosis in viral uptake. It also demonstrates that the expression of Nectin-1 and HVEM predominantly determines HSV-1 entry into HCjE cells. This is the first report of a cell type in which both Nectin-1 and HVEM are found critical for entry.
