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Yoshimi Takai - One of the best experts on this subject based on the ideXlab platform.
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Nectin 2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula
The Journal of Comparative Neurology, 2021Co-Authors: Hajime Shiotani, Muneaki Miyata, Kiyohito Mizutani, Kenji Mandai, Takeshi Kameyama, Miwako Yamasaki, Masahiko Watanabe, Yoshimi TakaiAbstract:The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule Nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named Nectin-2α spots, is not associated with the Nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and β-catenin. We showed here that Nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of Nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, β-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of Nectin-2 did not affect synapse formation in the IPN. These results indicate that Nectin-2α forms two types of adhesion apparatus in the MHb, namely Nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.
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Interaction of Nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula
Molecular and Cellular Neuroscience, 2018Co-Authors: Hajime Shiotani, Muneaki Miyata, Kiyohito Mizutani, Kenji Mandai, Akira Mizoguchi, Hideki Mochizuki, Shujie Wang, Yoshimi TakaiAbstract:Abstract The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule Nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas Nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of Nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how Nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that Nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with Nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of Nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that Nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.
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A Novel Subcellular Machine Contributes to Basal Junction Remodeling in the Seminiferous Epithelium
Biology of Reproduction, 2013Co-Authors: Jnelle S Young, Yoshimi Takai, Marc De Asis, Jane Cipollone, Calvin D. Roskelley, Peter K. Nicholls, Peter G. Stanton, Wanyin Deng, B. Brett FinlayAbstract:Tubulobulbar complexes are cytoskeleton-related membrane structures that develop at sites of intercellular attachment in mammalian seminiferous epithelium. At apical junctions between Sertoli cells and spermatids, the structures internalize adhesion junctions and are a component of the sperm release mechanism. Here we explore the possibility that tubulobulbar complexes that form at the blood-testis barrier are subcellular machines that internalize basal junction complexes. Using electron microscopy, we confirmed that morphologically identifiable tight and gap junctions are present in basal tubulobulbar complexes in rats. In addition, immunological probes for claudin-11 (CLDN11), connexin-43 (GJA1), and Nectin-2 (PVRL2) react with linear structures at the light level that we interpret as tubulobulbar complexes, and probes for early endosome antigen 1 (EEA1) and Rab5 (RAB5A) react in similar locations. Significantly, fluorescence patterns for actin and claudin-11 indicate that the amount of junction present is dramatically reduced over the time period that tubulobulbar complexes are known to be most prevalent during spermatogenesis. We also demonstrated, using electron microscopy and fluorescence microscopy, that tubulobulbar complexes develop at basal junctions in primary cultures of Sertoli cells and that like their in vivo counterparts, the structures contain junction proteins. We use this culture system together with transfection techniques to show that junction proteins from one transfected cell occur in structures that project into adjacent nontransfected cells as predicted by the junction internalization hypothesis. On the basis of our findings, we present a new model for basal junction remodeling as it relates to spermatocyte translocation in the seminiferous epithelium.
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internalization of adhesion junction proteins and their association with recycling endosome marker proteins in rat seminiferous epithelium
Reproduction, 2012Co-Authors: Jnelle S Young, Yoshimi Takai, Katarina L Kojic, Wayne A VoglAbstract:Tubulobulbar complexes (TBCs) are elaborate cytoskeleton-related structures that are formed in association with intercellular junctions in the seminiferous epithelium. They consist of a cylindrical double-membrane core composed of the plasma membranes of the two attached cells, cuffed by a dendritic network of actin filaments. TBCs are proposed to be subcellular machines that internalize intercellular junctions during the extensive junction remodeling that occurs during spermatogenesis. At the apical sites of attachment between Sertoli cells and spermatids, junction disassembly is part of the sperm release mechanism. In this study, we used immunological probes to explore junction internalization and recycling at apical TBCs in the rat seminiferous epithelium. We demonstrate that β1-integrin and Nectin 2 were concentrated at the ends of TBCs and for the first time show that the early endosome marker RAB5A was also distinctly localized at the ends of TBCs that appear to be the 'bulbar' regions of the complexes. Significantly, we also demonstrate that the 'long-loop' recycling endosome marker RAB11A was co-distributed with Nectin 2 at junctions with early spermatids deeper in the epithelium. Our results are consistent with the hypothesis that TBCs associated with late spermatids internalize adhesion junctions and also indicate that some of the internalized junction proteins may be recycled to form junctions with the next generation of spermatids.
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Role of cell adhesion molecule Nectin-3 in spermatid development
Genes to cells : devoted to molecular & cellular mechanisms, 2006Co-Authors: Maiko Inagaki, Kenji Irie, Hiroyoshi Ishizaki, Miki Tanaka-okamoto, Jun Miyoshi, Yoshimi TakaiAbstract:Seminiferous epithelia of the testes contain two types of intercellular junctions: Sertoli-Sertoli junctions and Sertoli-spermatid junctions. The former junctions are equipped with tight and adherens junctions while the latter junctions are not. Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, Nectin-2 and Nectin-3, asymmetrically localize at the Sertoli cell side and at the spermatid side of Sertoli-spermatid junctions, respectively. They heterophilically trans-interact to make contact between the two cells. Nectin-2(-/-) mice have shown male-specific infertility, disrupted Sertoli-spermatid junctions and morphologically impaired spermatid development. Here we report testicular phenotypes of Nectin-3(-/-) mice exhibiting male-specific infertility. Nectin-3(-/-) mice had defects in the later steps of sperm morphogenesis including distorted nuclei and abnormal distribution of mitochondria, as well as in localization of Nectin-2 at the Sertoli-spermatid junctions. Transplantation of wild-type spermatogenic stem cells into the Nectin-3(-/-) testes partially rescued these defects in sperm morphogenesis. These results indicate that the heterophilic trans-interaction between Nectin-2 and Nectin-3 is essential for the formation and maintenance of Sertoli-spermatid junctions that plays a critical role in spermatid development.
Patricia G. Spear - One of the best experts on this subject based on the ideXlab platform.
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mutations in herpes simplex virus glycoprotein d that prevent cell entry via Nectins and alter cell tropism
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sharmila Manoj, Miri Yoon, Cheryl R Jogger, Dawn Myscofski, Patricia G. SpearAbstract:Glycoprotein D (gD) determines which cells can be infected by herpes simplex virus (HSV) by binding to one of the several cell surface receptors that can mediate HSV entry or cell fusion. These receptors include the herpesvirus entry mediator (HVEM), Nectin-1, Nectin-2, and sites in heparan sulfate generated by specific 3-O-sulfotransferases. The objective of the present study was to identify residues in gD that are critical for physical and functional interactions with Nectin-1 and Nectin-2. We found that double or triple amino acid substitutions at positions 215, 222, and 223 in gD caused marked reduction in gD binding to Nectin-1 and a corresponding inability to function in cell fusion or entry of HSV via Nectin-1 or Nectin-2. These substitutions either enhanced or did not significantly inhibit functional interactions with HVEM and modified heparan sulfate. These and other results demonstrate that different domains of gD, with some overlap, are critical for functional interactions with each class of entry receptor. Viral entry assays, using gD mutants described here and previously, revealed that Nectins are the principal entry receptors for selected human cell lines of neuronal and epithelial origin, whereas HVEM or Nectins could be used to mediate entry into a T lymphocyte line. Because T cells and fibroblasts can be infected via HVEM, HSV strains carrying gD mutations that prevent entry via Nectins may establish transient infections in humans, but perhaps not latent infections of neurons, and are therefore candidates for development of safe live virus vaccines and vaccine vectors.
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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.
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differences in the n termini of herpes simplex virus type 1 and 2 gds that influence functional interactions with the human entry receptor Nectin 2 and an entry receptor expressed in chinese hamster ovary cells
Journal of Virology, 2003Co-Authors: Anna Zago, Patricia G. SpearAbstract:Amino acid differences at seven positions in the N termini of the glycoproteins D (gDs) specified by herpes simplex virus type 1 (HSV-1) and HSV-2 are largely responsible for the significantly higher cell fusion activity of HSV-2 gD with Chinese hamster ovary cells expressing human Nectin-2 or only an endogenous hamster receptor.
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Mutations in the N-Terminal Domains of Nectin-1 and Nectin-2 Reveal Differences in Requirements for Entry of Various Alphaherpesviruses and for Nectin-Nectin Interactions
Journal of virology, 2002Co-Authors: Frank Struyf, Wanda M. Martinez, Patricia G. SpearAbstract:Nectin-1 and Nectin-2 are closely related cell surface molecules of the immunoglobulin superfamily and are expressed in a variety of cell types. To date, four members of the Nectin family have been described, each with multiple isoforms (10, 23, 31, 32, 34), and all related to the poliovirus receptor CD155 (26). Most members of the Nectin family are membrane-bound proteins with an extracellular N-terminal variable region-like (V-like) domain, two extracellular constant region-like (C-like) domains, a transmembrane region, and a cytoplasmic tail; however, a soluble isoform of Nectin-1 has also been described (21). Nectin-1 and Nectin-2, but not Nectin-3 or Nectin-4, can function with different specificities as entry receptors for mammalian alphaherpesviruses (5, 14, 22, 39). After initial binding of the virion via viral glycoprotein B (gB) or gC to heparan sulfate on the cell surface, Nectin-1 or Nectin-2 can mediate viral entry through interaction with viral gD, followed by fusion of the viral envelope with the cellular plasma membrane. The amino-terminal V-like domain has the critical determinants for binding to gD (4, 17, 20) and for viral entry (3, 4, 12, 25). Nectin-1 and Nectin-2 have different specificities for wild-type alphaherpesviruses. While Nectin-1 shows activity as a receptor for herpes simplex virus type 1 (HSV-1), HSV-2, pseudorabies virus (PRV), and bovine herpesvirus type 1 (BHV-1) (5, 14), Nectin-2 mediates entry of HSV-2 and PRV but not that of HSV-1 or BHV-1 (39). Mutant strains of HSV-1 that have single amino acid substitutions at position 25 or 27 in gD (2, 7) can enter cells via both Nectin-1 and Nectin-2 (14, 22, 39). Nectins and associated cytoplasmic proteins are components of cadherin-based adherens junctions in both epithelial and nonepithelial cells (37, 38). Some membrane-bound isoforms of the Nectins contain in their cytoplasmic tail a carboxy-terminal PDZ-binding domain through which they can bind afadin, which itself binds to F-actin and α-catenin (28, 30). In the presence or absence of cadherin, Nectin-1 and Nectin-2 can engage in homotypic cis and trans interactions (28). Both molecules also show heterotypic trans interactions with other members of the Nectin family. Nectin-1 and Nectin-2 can bind to Nectin-3 (31, 34), and Nectin-1 binds to Nectin-4 (32). It appears that, for both Nectin-1 and Nectin-2, the V-like domain is important for viral entry (3-5, 12, 20, 24, 25) and Nectin-Nectin interactions (11, 18, 28, 32) and that regions critical for both activities may overlap. Also, soluble forms of viral gD were able to block cell adhesion mediated by Nectin-1 homotypic trans interactions (18, 33) and to partially inhibit the binding of soluble forms of Nectin-3 and Nectin-4 to Nectin-1 (11). By use of hybrid molecules constructed from members of the Nectin-CD155 family that differ in functional activities, it has been shown that regions critical to HSV entry are located in loops bounding the predicted C′ beta strand in the V domain of both Nectin-1 and Nectin-2 (3, 24, 25) and that regions critical to heterotypic trans interactions are located within a region including the C-C′-C"-D beta strands in the V domain of Nectin-1 (11) (Fig. (Fig.1).1). For both HSV entry and heterotypic trans interactions, numerous amino acid substitutions within the V domain but outside of these critical regions are tolerated. Mutations in one particular region of the V domains of both Nectin-1 and Nectin-2 have been shown to significantly reduce HSV entry activity (24, 25) but have not been tested for effects on trans interactions between Nectins. A mutation in a different highly conserved region of the V-like domain of mouse Nectin-2 has been shown to prevent homotypic trans interactions (28), but no information on viral entry activity was presented. In this study, we aimed to clarify the requirements for human Nectin-1 and Nectin-2 engagement in homotypic and heterotypic trans interactions and mediation of the entry of various alphaherpesviruses. We constructed Nectin-1 and Nectin-2 mutants with single amino acid changes in the V-like domain of the molecules on the basis of information from previous studies of mutations that were shown to have deleterious effects on HSV entry or homotypic trans interactions and on a Nectin-2 V domain polymorphism reported in a public database. Viral entry properties, gD binding, Nectin-3 binding, cellular localization indicative of homotypic trans interactions, and colocalization with gD were examined by expressing the receptor molecules in Chinese hamster ovary (CHO) cells, which normally lack alphaherpesvirus entry receptors. The results show that the structural requirements of the Nectin activities examined partially overlap but differ. FIG. 1. Alignment of amino acid sequences in the N-terminal V-like domains of human Nectin-1 (hNectin-1), human Nectin-2 (hNectin-2), and mouse Nectin-2 (mNectin-2). The proposed location of beta-strands (labeled A to G), based on a model of the poliovirus receptor ... (Parts of this work were presented at the American Society for Virology 20th Annual Meeting, Madison, Wis., 21 to 25 July 2001, and the 26th International Herpesvirus Workshop, Regensburg, Germany, 28 July to 3 August 2001.)
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Search for Polymorphisms in the Genes for Herpesvirus Entry Mediator, Nectin-1, and Nectin-2 in Immune Seronegative Individuals
The Journal of infectious diseases, 2001Co-Authors: Frank Struyf, Christine M. Posavad, Els Keyaerts, Marc Van Ranst, Lawrence Corey, Patricia G. SpearAbstract:Recently, individuals have been identified who possess T cell responses to herpes simplex virus (HSV) antigens despite the absence of detectable anti-HSV antibodies in their serum. The significance of this immune seronegative status is unclear, but it could indicate resistance to overt HSV infection. The aims of the present study were to investigate whether genetic differences in receptors used by HSV for cell entry (herpesvirus entry mediator [HVEM], Nectin-1, and Nectin-2) could be detected in immune seronegative individuals. Coding polymorphisms were identified in the HVEM and Nectin-1 genes. The variant receptor proteins were expressed, and their ability to bind the viral ligand glycoprotein D and to mediate HSV entry after transient transfection into normally resistant cells was compared with that of their wild-type counterparts. HSV entry activity in wild-type and variant forms of the receptors was indistinguishable, which indicates that the polymorphisms observed are unlikely to explain the possible restrictions on HSV replication or spread in immune seronegative individuals.
Anthony V Nicola - One of the best experts on this subject based on the ideXlab platform.
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structure function analysis of herpes simplex virus glycoprotein b with fusion from without activity
Virology, 2008Co-Authors: Devin G Roller, Stephen J Dollery, James L Doyle, Anthony V NicolaAbstract:Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein synthesis. The combination of two amino acid mutations in envelope glycoprotein B (gB), one in the ectodomain and one in the cytoplasmic tail, can confer FFWO activity to wild type herpes simplex virus (HSV). In this report, we analyzed the entry and cell fusion phenotypes of HSV that contains FFWO gB, with emphasis on the cellular receptors for HSV, Nectin-1, Nectin-2 and HVEM. The ability of an HSV strain with FFWO gB to efficiently mediate FFWO via a specific gD-receptor correlated with its ability to mediate viral entry by that receptor. A FFWO form of gB was not sufficient to switch the entry of HSV from a pH-dependent, endocytic pathway to a direct fusion, pH-independent pathway. The conformation of gB with FFWO activity was not globally altered relative to wild type. However, distinct monoclonal antibodies had reduced reactivity with FFWO gB, suggesting an altered antigenic structure relative to wild type. FFWO was blocked by preincubation of virions with neutralizing antibodies to gB or gD. Together with previous studies, the results indicate that the roles of gB in FFWO and in virus-cell fusion during entry are related but not identical. This study also suggests that the FFWO function of gB is not a specific determinant for the selection of HSV entry pathway and that antigenic differences in FFWO gB may reflect its enhanced fusion activity.
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Nectin 2 mediated entry of a syncytial strain of herpes simplex virus via ph independent fusion with the plasma membrane of chinese hamster ovary cells
Virology Journal, 2006Co-Authors: Mark G Delboy, Jennifer L Patterson, Aimee M Hollander, Anthony V NicolaAbstract:Background Herpes simplex virus (HSV) can utilize multiple pathways to enter host cells. The factors that determine which route is taken are not clear. Chinese hamster ovary (CHO) cells that express glycoprotein D (gD)-binding receptors are model cells that support a pH-dependent, endocytic entry pathway for all HSV strains tested to date. Fusion-from-without (FFWO) is the induction of target cell fusion by addition of intact virions to cell monolayers in the absence of viral protein expression. The receptor requirements for HSV-induced FFWO are not known. We used the syncytial HSV-1 strain ANG path as a tool to evaluate the complex interplay between receptor usage, membrane fusion, and selection of entry pathway.
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roles for endocytosis and low ph in herpes simplex virus entry into hela and chinese hamster ovary cells
Journal of Virology, 2003Co-Authors: Anthony V Nicola, Anna M Mcevoy, Sharon E StrausAbstract:Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, Nectin-1, or Nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.
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roles for endocytosis and low ph in herpes simplex virus entry into hela and chinese hamster ovary cells
Journal of Virology, 2003Co-Authors: Anthony V Nicola, Anna M Mcevoy, Sharon E StrausAbstract:Many viruses utilize the cellular endocytic machinery to transport their genetic material to the cell interior. In addition to depending on this machinery, enveloped viruses such as influenza virus and nonenveloped viruses such as rhinovirus 2 require an acid pH for escape from the endocytic pathway to the host cytosol (35). For some enveloped viruses, the low-pH environment of the endosome triggers fusion of the virion envelope with cellular membranes. For other viruses, endocytic entry is not followed by a pH-triggered step. For example, Epstein-Barr virus and duck hepatitis B virus enter certain types of cells by endocytosis yet they do not require exposure to a low pH to penetrate the cytosol (27, 38). By contrast, viruses such as human immunodeficiency virus (HIV) and Sendai virus usually fuse with the plasma membrane in a pH-independent manner without a requirement for endocytosis (19). Entry of herpes simplex virus type 1 (HSV-1) and HSV-2 is initiated by interaction of virions with cell surface glycosaminoglycans (48). This is followed by binding to one of several entry receptors utilized by HSV, including HveA (herpesvirus entry mediator A, also known as HVEM), a member of the tumor necrosis factor receptor family (39), and the immunoglobulin-like intercellular adhesion molecules Nectin-1 (10, 18, 33, 55) and Nectin-2 (32, 58), also known as HveC/HIgR/PRR1 and HveB/PRR2, respectively. Chinese hamster ovary (CHO) cells are naturally resistant to HSV entry and require expression of a receptor to remove the block to entry (39, 47). Thus, they have been used as a model system in which to study receptor-mediated entry of HSV. HSV envelope glycoprotein gD binds to all known alphaherpesvirus receptors (8, 53), and this interaction is essential for entry (25). Fusion of bound virus with host membranes is thought to be triggered by the interaction with gD receptors. Studies of HSV-infected cells at early times postinfection using electron microscopy (EM) have detected virions fusing with the plasma membrane (16, 17, 34, 40, 50, 51), as well as virions inside membrane-bound vesicles (6, 11, 13, 17, 23, 40, 51). On the basis of morphological analyses alone, it is not possible to assess whether any given virion is engaging a pathway that could lead to infection. Virions are often detected in vesicles during nonproductive infections in which either the cells do not support virus penetration (6) or the virus itself is entry defective (61). In such instances, it is thought that the internalized virions are targeted ultimately for degradation. EM, coupled with treatments with a neutralizing antibody, has been used to demonstrate that infectious entry of HSV into Vero and HEp-2 cells could occur via fusion at the plasma membrane (16, 17). However, successful penetration of HSV from an intracellular compartment following endocytosis has not been excluded. Weak bases, such as chloroquine, that block endosomal acidification and entry of viruses that require a low pH were shown to have little effect on early events in HSV infection of HEp-2 and Vero cells (60). Largely on the basis of the study of these cell types, it is thought that HSV cell entry does not require a low pH. The pathway utilized by HSV for entry into two other common cultured cell lines was investigated. The conclusions obtained were different from those of earlier studies. On the basis of several experimental approaches, evidence is provided that CHO-K1 cells expressing HSV receptors and HeLa cells support an endocytic, pH-dependent route for HSV entry. Thus, HSV infection can be initiated by more than one pathway in a cell-specific manner.
Roselyn J Eisenberg - One of the best experts on this subject based on the ideXlab platform.
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Comparative usage of herpesvirus entry mediator A and Nectin-1 by laboratory strains and clinical isolates of herpes simplex virus.
Virology, 2004Co-Authors: Claude Krummenacher, Gary H Cohen, Isabelle Baribaud, J. Charles Whitbeck, Frédéric Baribaud, Manuel Ponce De Leon, Roselyn J EisenbergAbstract:The herpesvirus entry mediator A (HVEM/HveA) and Nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use Nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and Nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and Nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of Nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or Nectin-2. Finally, we compared the ability of HVEM and Nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that Nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.
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structure based mutagenesis of herpes simplex virus glycoprotein d defines three critical regions at the gd hvea hvem binding interface
Journal of Virology, 2003Co-Authors: Sarah A Connolly, Gary H Cohen, Daniel J Landsburg, Andrea Carfi, Don C Wiley, Roselyn J EisenbergAbstract:Herpes simplex virus (HSV) entry into cells requires the binding of glycoprotein D (gD) to one of several cell surface receptors. The crystal structure of gD bound to one of these receptors, HveA/HVEM, reveals that the core of gD comprises an immunoglobulin fold flanked by a long C-terminal extension and an N-terminal hairpin loop. HveA is a member of the tumor necrosis factor receptor family and contains four cysteine-rich domains (CRDs) characteristic of this family. Fourteen amino acids within the gD N-terminal loop comprise the entire binding site for HveA. To determine the contribution of each gD contact residue to virus entry, we constructed gD molecules mutated in these amino acids. We determined the abilities of the gD mutants to bind receptors, facilitate virus entry, and mediate cell-cell fusion. Seven of the gD mutants exhibited wild-type levels of receptor binding and gD function. Results from the other seven gD mutants revealed three critical regions at the gD-HveA interface. (i) Several gD residues that participate in an intermolecular β-sheet with HveA were found to be crucial for HveA binding and entry into HveA-expressing cells. (ii) Two gD residues that contact HveA-Y23 contributed to HveA binding but were not required for mediating entry into cells. HveA-Y23 fits into a crevice on the surface of gD and was previously shown to be essential for gD binding. (iii) CRD2 was previously shown to contribute to gD binding, and this study shows that one gD residue that contacts CRD2 contributes to HveA binding. None of the gD mutations prevented interaction with Nectin-1, another gD receptor. However, when cotransfected with the other glycoproteins required for fusion, two gD mutants gained the ability to mediate fusion of cells expressing Nectin-2, a gD receptor that interacts with several laboratory-derived gD mutants but not with wild-type gD. Thus, results from this panel of gD mutants as well as those of previous studies (A. Carfi, S. H. Willis, J. C. Whitbeck, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and D. C. Wiley, Mol. Cell 8:169-179, 2001, and S. A. Connolly, D. J. Landsburg, A. Carfi, D. C. Wiley, R. J. Eisenberg, and G. H. Cohen, J. Virol. 76:10894-10904, 2002) provide a detailed picture of the gD-HveA interface and the contacts required for functional interaction. The results demonstrate that of the 35 gD and HveA contact residues that comprise the gD-HveA interface, only a handful are critical for complex formation.
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glycoprotein d homologs in herpes simplex virus type 1 pseudorabies virus and bovine herpes virus type 1 bind directly to human hvec Nectin 1 with different affinities
Virology, 2001Co-Authors: Sarah A Connolly, Claude Krummenacher, Gary H Cohen, Charles J Whitbeck, Ann H Rux, Sylvia Van Drunen Littelvan Den Hurk, Roselyn J EisenbergAbstract:Distinct subsets of human receptors for alphaherpesviruses mediate the entry of herpes simplex virus (HSV), pseudorabies virus (PrV), or bovine herpes virus type 1 (BHV-1) into cells. Glycoprotein D (gD) is essential for receptor-mediated entry of all three viruses into cells. However, the gD homologs of these viruses share only 22-33% amino acid identity. Several entry receptors for HSV have been identified. Two of these, HveA (HVEM) and HveC (Nectin-1), mediate entry of most HSV-1 and HSV-2 strains and are bound directly by HSV gD. A third receptor, HveB (Nectin-2), mediates entry of HSV-2 and only a limited number of HSV-1 strains. HveB and HveC can also serve as entry receptors for PrV, whereas only HveC can serve this function for BHV-1. We show here that gD from PrV and BHV-1 binds directly to the human receptors that mediate PrV and BHV-1 entry. We expressed soluble forms of PrV gD and BHV-1 gD using recombinant baculoviruses and purified each protein. Using ELISA, we detected direct binding of PrV gD to HveB and HveC and direct binding of BHV-1 gD to HveC. Biosensor analysis revealed that PrV gD had a 10-fold higher affinity than HSV-1 gD for human HveC. In contrast, the binding of BHV-1 gD to HveC was weak. PrV gD and HSV-1 gD competed for binding to the V domain of HveC and both inhibited entry of the homologous and heterologous viruses. These data suggest that the two forms of gD bind to a common region on human HveC despite their low amino acid similarity. Based on affinities for human HveC, we predict a porcine HveC homolog may be important for PrV infection in its natural host, whereas a BHV-1 infection in its natural host may be mediated by a receptor other than a bovine HveC homolog.
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structural basis for cd96 immune receptor recognition of Nectin like protein 5 cd155
Structure, 2019Co-Authors: Felix A Deuss, Gabrielle M Watson, Jamie Rossjohn, Richard BerryAbstract:Summary CD96, DNAM-1, and TIGIT constitute a group of immunoglobulin superfamily receptors that are key regulators of tumor immune surveillance. Within this axis, CD96 recognizes the adhesion molecule Nectin-like protein-5 (necl-5), although the molecular basis underpinning this interaction remains unclear. We show that the first immunoglobulin domain (D1) of CD96 is sufficient to mediate a robust interaction with necl-5, but not the DNAM-1 and TIGIT ligand, Nectin-2. The crystal structure of CD96-D1 bound to the necl-5 ectodomain revealed that CD96 recognized necl-5 D1 via a conserved “lock-and-key” interaction observed across TIGIT:necl complexes. Specific necl-5 recognition was underpinned by a novel structural motif within CD96, namely an “ancillary key”. Mutational analysis showed that this specific residue was critical for necl-5 binding, while simultaneously providing insights into the unique ligand specificity of CD96.
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recognition of Nectin 2 by the natural killer cell receptor t cell immunoglobulin and itim domain tigit
Journal of Biological Chemistry, 2017Co-Authors: Felix A Deuss, Jamie Rossjohn, Benjamin S Gully, Richard BerryAbstract:T cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed on the surface of natural killer (NK) cells. TIGIT recognizes Nectin and Nectin-like adhesion molecules and thus plays a critical role in the innate immune response to malignant transformation. Although the TIGIT Nectin-like protein-5 (necl-5) interaction is well understood, how TIGIT engages Nectin-2, a receptor that is broadly over-expressed in breast and ovarian cancer, remains unknown. Here, we show that TIGIT bound to the immunoglobulin domain of Nectin-2 that is most distal from the membrane with an affinity of 6 μm, which was moderately lower than the affinity observed for the TIGIT/necl-5 interaction (3.2 μm). The TIGIT/Nectin-2 binding disrupted pre-assembled Nectin-2 oligomers, suggesting that receptor-ligand and ligand-ligand associations are mutually exclusive events. Indeed, the crystal structure of TIGIT bound to the first immunoglobulin domain of Nectin-2 indicated that the receptor and ligand dock using the same molecular surface and a conserved “lock and key” binding motifs previously observed to mediate Nectin/Nectin homotypic interactions as well as TIGIT/necl-5 recognition. Using a mutagenesis approach, we dissected the energetic basis for the TIGIT/Nectin-2 interaction and revealed that an “aromatic key” of Nectin-2 is critical for this interaction, whereas variations in the lock were tolerated. Moreover, we found that the C-C′ loop of the ligand dictates the TIGIT binding hierarchy. Altogether, these findings broaden our understanding of Nectin/Nectin receptor interactions and have implications for better understanding the molecular basis for autoimmune disease and cancer.
