Trypanosoma cruzi invade a mammalian epithelial cell in a polarized manner

We have determined that parasite entry into host cells can be influenced by cell polarity using a DNA probe to quantitate the infection of cultured Madin-Darby canine kidney (MDCK) epithelial cells by Trypanosoma cruzi, the agent of Chagas' disease. Confluent MDCK cells are polarized, with their plasma membrane separated by tight junctions into two domains, apical and basolateral. We show that T. cruzi forms corresponding to the insect infective stages (metacyclics) and the vertebrate blood stages (trypomastigotes) enter confluent MDCK cells preferentially through their basolateral domains. Sparsely plated MDCK cells are less polarized and are better infected than confluent cells. Scanning electron microscopy showed that 92% +/- 4% of the parasites entered at the edges of cells.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry

Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor.     

Basic science track. Entry and release of canine coronavirus from polarized epithelial cells

Virus entry into and release from epithelial cells are polarized as a result of the distribution of the viral receptors. In order to establish the polarity of entry and release of CCoV from epithelial cells, the interactions of the virus with A72 and CrFK cells grown on permeable supports was evaluated, and the amount of infective virus in the apical and in the basolateral media was determined and compared. Infection of A72 cells after different times post seeding demonstrated that CCoV grow after infection from both apical and basolateral sides. In CrFK cells, CCoV was observed in both compartments only in the later phase of the infection. To establish the reciprocal binding of CCoV on plasma membrane, A72 cells on a permeable support were preincubated with a mAb specific for CCoV. Infection from the apical side was blocked by mAb applied to that side; in contrast, such treatment on the basolateral side had no effect on the infectious process. Similarly, the low levels of CCoV observed after basolateral exposure to virus was abolished following mAb treatment of that side. The identification of CCoV into the basolateral medium could play an important role in the viral pathogenesis.     

Epstein-Barr Virus Transcytosis through Polarized Oral Epithelial Cells

Although Epstein-Barr virus (EBV) is an orally transmitted virus, viral transmission through the oropharyngeal mucosal epithelium is not well understood. In this study, we investigated how EBV traverses polarized human oral epithelial cells without causing productive infection. We found that EBV may be transcytosed through oral epithelial cells bidirectionally, from both the apical to the basolateral membranes and the basolateral to the apical membranes. Apical to basolateral EBV transcytosis was substantially reduced by amiloride, an inhibitor of macropinocytosis. Electron microscopy showed that virions were surrounded by apical surface protrusions and that virus was present in subapical vesicles. Inactivation of signaling molecules critical for macropinocytosis, including phosphatidylinositol 3-kinases, myosin light-chain kinase, Ras-related C3 botulinum toxin substrate 1, p21-activated kinase 1, ADP-ribosylation factor 6, and cell division control protein 42 homolog, led to significant reduction in EBV apical to basolateral transcytosis. In contrast, basolateral to apical EBV transcytosis was substantially reduced by nystatin, an inhibitor of caveolin-mediated virus entry. Caveolae were detected in the basolateral membranes of polarized human oral epithelial cells, and virions were detected in caveosome-like endosomes. Methyl β-cyclodextrin, an inhibitor of caveola formation, reduced EBV basolateral entry. EBV virions transcytosed in either direction were able to infect B lymphocytes. Together, these data show that EBV transmigrates across oral epithelial cells by (i) apical to basolateral transcytosis, potentially contributing to initial EBV penetration that leads to systemic infection, and (ii) basolateral to apical transcytosis, which may enable EBV secretion into saliva in EBV-infected individuals.     

Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.     

Apical entry and release of severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial cells

Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (CoV) known as SARS-CoV, is a contagious and life-threatening respiratory illness with pneumocytes as its main target. A full understanding of how SARS-CoV would interact with lung epithelial cells will be vital for advancing our knowledge of SARS pathogenesis. However, an in vitro model of SARS-CoV infection using relevant lung epithelial cells is not yet available, making it difficult to dissect the pathogenesis of SARS-CoV in the lungs. Here, we report that SARS-CoV can productively infect human bronchial epithelial Calu-3 cells, causing cytopathic effects, a process reflective of its natural course of infection in the lungs. Indirect immunofluorescence studies revealed a preferential expression of angiotensin-converting enzyme 2 (ACE-2), the functional receptor of SARS-CoV, on the apical surface. Importantly, both ACE-2 and viral antigen appeared to preferentially colocalize at the apical domain of infected cells. In highly polarized Calu-3 cells grown on the membrane inserts, we found that cells exposed to virus through the apical rather than the basolateral surface showed high levels of viral replication. Progeny virus was released into the apical chamber at titers up to 5 logs higher than those recovered from the basolateral chambers of polarized cultures. Taken together, these results indicate that SARS-CoV almost exclusively entered and was released from the apical domain of polarized Calu-3 cells, which might provide important insight into the mechanism of transmission and pathogenesis of SARS-CoV.     

Morphological and functional polarity of an epithelial thyroid cell line

The thyroid epithelial cell line FRT in monolayer culture appeared to be strongly polarized by morphological criteria. Cells were connected by tight junctions, exposed microvilli toward the culture medium and formed domes at confluency. FRT cells were infected with vesicular stomatitis virus (VSV) and Sindbis virus and the budding polarity was examined 8 and 16 h after infection, respectively. VSV budding occurred preferentially from the basolateral domain of plasma membrane, while Sindbis virus budding was mostly apical. The distribution of VSV and Sindbis virus glycoproteins, as determined by the immuno-gold technique, correlated well with the budding polarity. Polarized budding was not observed in isolated cells in suspension.     

Human cytomegalovirus infection of caco-2 cells occurs at the basolateral membrane and is differentiation state dependent

Epithelial cells are known to be a major target for human cytomegalovirus (HCMV) infection; however, the analysis of virus-cell interactions has been difficult to approach due to the lack of in vitro models. In this study, we established a polarized epithelial cell model using a colon epithelial cell-derived cell line (Caco-2) that is susceptible to HCMV infection at early stages of cellular differentiation. Infection of polarized cells was restricted to the basolateral surface whereas virus was released apically, which was consistent with the apical and not basolateral surface localization of two essential viral glycoproteins, gB and gH. HCMV infection resulted in the development of a cytopathology characteristic of HCMV infection of colon epithelium in vivo, and infection did not spread from cell to cell. The inability of HCMV to infect Caco-2 cells at late stages of differentiation was due to a restriction at the level of viral entry and was consistent with the sequestration of a cellular receptor for HCMV. These observations provide the first evidence that restriction of HCMV replication in epithelial cells is due to a receptor-mediated phenomenon.     

Entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells.

The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract of pigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcine epithelial cell line. To determine the site of viral entry, LLC-PK1 cells were grown on permeable filter supports and infected with TGEV from the apical or basolateral side. Initially after plating, the virus was found to enter the cells from both sides. During further development of cell polarity, however, the entry became restricted to the apical membrane. Viral entry could be blocked by a monoclonal antibody to the viral receptor aminopeptidase N. Confocal laser scanning microscopy showed that this receptor protein was present at both the apical and basolateral plasma membrane domains just after plating of the cells but that it became restricted to the apical plasma membrane during culture. To establish the site of viral release, the viral content of the apical and basolateral media of apically infected LLC-PK1 cells was measured by determining the amount of radioactively labelled viral proteins and infectious viral particles. We found that TGEV was preferentially released from the apical plasma membrane. This conclusion was confirmed by electron microscopy, which demonstrated that newly synthesized viral particles attached to the apical membrane. The results support the idea that the rapid lateral spread of TGEV infection over the intestinal epithelia occurs by the preferential release of virus from infected epithelial cells into the gut lumen followed by efficient infection of nearby cells through the apical domain.     

Hepatocyte growth factor stimulates adenoviral-mediated gene transfer across the apical membrane of epithelial cells

BACKGROUND: The apical surface of polarized epithelial cells is relatively resistant to gene delivery by various agents including adenoviral vectors. Hepatocyte growth factor (HGF) dedifferentiates previously well-polarized Madin-Darby canine kidney (MDCK) cell monolayers by altering cell-surface polarity and inhibiting tight junction function. METHODS: We used an in vitro model of polarized MDCK cells grown on permeable supports to examine the effects of HGF pretreatment on adenoviral (Ad)-mediated gene delivery through the apical surface of epithelial cell monolayers. RESULTS: HGF pretreatment of MDCK cell monolayers for 72 h increased Ad-mediated gene transfer and expression of enhanced green fluorescent protein (EGFP) and luciferase in a dose-dependent fashion. Time-course analysis of HGF-induced stimulation of Ad-mediated gene transfer was seen after 24 h and increased further with pretreatment periods extending to 72 h. HGF pretreatment increased Ad-mediated gene transfer at varying multiplicity of infection (MOI; ranging from 0.2-2000). PCR analysis for adenoviral DNA in control and HGF-pretreated MDCK cells suggested increased entry of viral constructs into HGF-pretreated MDCK cell monolayers. HGF-induced alterations in cell polarity are reversible upon removal of HGF. CONCLUSIONS: These data demonstrate that HGF pretreatment of MDCK cells increases the sensitivity of the cells to Ad-mediated gene delivery. The mechanism by which this occurs appears to be through increased entry of adenovirus into epithelial cells. These data provide evidence that biological agents that transiently alter epithelial cell polarity and tight junction function can be used to augment Ad-mediated gene delivery into epithelial cells from the apical surface.     

Polarized sorting and trafficking in epithelial cells

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.     

Human coronavirus 229E infects polarized airway epithelia from the apical surface

Gene transfer to differentiated airway epithelia with existing viral vectors is very inefficient when they are applied to the apical surface. This largely reflects the polarized distribution of receptors on the basolateral surface. To identify new receptor-ligand interactions that might be used to redirect vectors to the apical surface, we investigated the process of infection of airway epithelial cells by human coronavirus 229E (HCoV-229E), a common cause of respiratory tract infections. Using immunohistochemistry, we found the receptor for HCoV-229E (CD13 or aminopeptidase N) localized mainly to the apical surface of airway epithelia. When HCoV-229E was applied to the apical or basolateral surface of well-differentiated primary cultures of human airway epithelia, infection primarily occurred from the apical side. Similar results were noted when the virus was applied to cultured human tracheal explants. Newly synthesized virions were released mainly to the apical side. Thus, HCoV-229E preferentially infects human airway epithelia from the apical surface. The spike glycoprotein that mediates HCoV-229E binding and fusion to CD13 is a candidate for pseudotyping retroviral envelopes or modifying other viral vectors.     

Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1(123)), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1(123), approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1(123) did not associate with the cell surface. sNec1(123)-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.     

Vaccinia virus preferentially enters polarized epithelial cells through the basolateral surface.

The uptake of vaccinia virus in polarized epithelial cells was studied to determine whether the site of entry was confined to either the apical or the basolateral membrane. Virus infection was monitored with a recombinant vaccinia virus carrying the luciferase reporter gene. Using cell lines MDCK and MDCK-D11, a clonal line with high transepithelial electrical resistance, we determined that vaccinia virus preferentially enters through the basolateral membrane. The possibility that there is a polarized cell surface distribution of vaccinia virus receptors which may be involved in systemic poxvirus infections is discussed.     

Cellular localization of nectin-1 and glycoprotein D during herpes simplex virus infection

During viral entry, herpes simplex virus (HSV) glycoprotein D (gD) interacts with a specific cellular receptor such as nectin-1 (PRR1/HveC/CD111) or the herpesvirus entry mediator A (HVEM/HveA). Nectin-1 is involved in cell-to-cell adhesion. It is located at adherens junctions, where it bridges cells through homophilic or heterophilic interactions with other nectins. Binding of HSV gD prevents nectin-1-mediated cell aggregation. Since HSV gD affects the natural function of nectin-1, we further investigated the effects of gD expression on nectin-1 during HSV infection or in transfected cells. We also studied the importance of the interaction between nectin-1 and the cytoplasmic protein afadin for HSV entry and spread as well as the effects of infection on this interaction. In these investigations, we used a panel of cells expressing nectin-1 or nectin-1-green fluorescent protein fusions as the only mediators of HSV entry. During HSV infection, nectin-1 localization at adherens junction was dramatically altered in a manner dependent on gD expression. Nectin-1 and gD colocalized at cell contact areas between infected and noninfected cells and at the edges of plaques. This specific accumulation of gD at junctions was driven by expression of nectin-1 in trans on the surface of adjacent cells. Reciprocally, nectin-1 was maintained at junctions by the trans expression of gD in the absence of a cellular natural ligand. Our observations indicate that newly synthesized gD substitutes for nectin-1 of infected cells at junctions with noninfected cells. We propose that gD attracts and maintains the receptor at junctions where it can be used for virus spread.     

Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells.

Respiratory syncytial (RS) virus infects the epithelium of the respiratory tract. We examined the replication and maturation of RS virus in two polarized epithelial cell lines, Vero C1008 and MDCK. Electron microscopy of RS virus-infected Vero C1008 cells revealed the presence of pleomorphic viral particles budding exclusively from the apical surface, often in clusters. The predominant type of particle was filamentous, 80 to 100 nm in diameter, and 4 to 8 microns in length, and evidence from filtration studies indicated that the filamentous particles were infectious. Cytopathology produced by RS virus infection of polarized Vero C1008 cells was minimal, and syncytia were not observed, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus. Infectivity assays with MDCK cells confirmed that in this cell line, RS virus was released into the apical medium but not into the basolateral medium. In addition, the majority of the RS virus transmembrane fusion glycoprotein on the cell surface was localized to the apical surface of the Vero C1008 cells. Taken together, these results demonstrate that RS virus matures at the apical surface of polarized epithelial cell lines.     

Structural features of nectin-2 (HveB) required for herpes simplex virus entry

One step in the process of herpes simplex virus (HSV) entry into cells is the binding of viral glycoprotein D (gD) to a cellular receptor. Human nectin-2 (also known as HveB and Prr2), a member of the immunoglobulin (Ig) superfamily, serves as a gD receptor for the entry of HSV-2, variant forms of HSV-1 that have amino acid substitutions at position 25 or 27 of gD (for example, HSV-1/Rid), and porcine pseudorabies virus (PRV). The gD binding region of nectin-2 is believed to be localized to the N-terminal variable-like (V) Ig domain. In order to identify specific amino acid sequences in nectin-2 that are important for HSV entry activity, chimeric molecules were constructed by exchange of sequences between human nectin-2 and its mouse homolog, mouse nectin-2, which mediates entry of PRV but not HSV-1 or HSV-2. The nectin-2 chimeric molecules were expressed in Chinese hamster ovary cells, which normally lack a gD receptor, and tested for cell surface expression and viral entry activity. As expected, chimeric molecules containing the V domain of human nectin-2 exhibited HSV entry activity. Replacement of either of two small regions in the V domain of mouse nectin-2 with amino acids from the equivalent positions in human nectin-2 (amino acids 75 to 81 or 89) transferred HSV-1/Rid entry activity to mouse nectin-2. The resulting chimeras also exhibited enhanced HSV-2 entry activity and gained the ability to mediate wild-type HSV-1 entry. Replacement of amino acid 89 of human nectin-2 with the corresponding mouse amino acid (M89F) eliminated HSV entry activity. These results identify two different amino acid sequences, predicted to lie adjacent to the C' and C" beta-strands of the V domain, that are critical for HSV entry activity. This region is homologous to the human immunodeficiency virus binding region of CD4 and to the poliovirus binding region of CD155.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1

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 Å 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.

Authors Summary

Herpes simplex virus (HSV) is a widespread human pathogen. Four viral glycoproteins (gD, gB, gH/gL) are required for HSV entry into host cells. gD binding to a cell surface receptor triggers conformational changes in the other viral glycoproteins leading to membrane fusion and viral entry. Two structurally unrelated cellular protein receptors, nectin-1 and HVEM, can mediate HSV entry upon binding to gD. The structure presented here reveals the molecular basis for the stable interaction between HSV-1 gD and the receptor nectin-1. Comparison with the previously determined structures of the gD/HVEM complex and unliganded gD shows that, despite the fact that the two receptors interact with different binding sites, they both cause a similar conformational change in gD. Therefore, our data point to a conserved mechanism for receptor mediated activation of the HSV entry process. In addition, the gD/Nectin-1 structure reveals that the gD-binding site overlaps with a surface involved in nectin-1 homo-dimerization. This observation explains how gD interferes with the cell adhesion function of nectin-1. Finally, the gD/Nectin-1 complex displays similarities with other viral ligands bound to immunoglobulin-like receptors suggesting a convergent mechanism for receptors selection and usage.