Binding Of gD Research Articles

Functional identification of Gd 3+ binding site of metabotropic glutamate receptor 1α

We previously reported that the metabotropic glutamate receptor1α (mGluR1α) has a sensitivity to extracellular polyvalent cations such as Ca 2+ and Gd 3+ as well as glutamate. Gd 3+ binding site was recently identified by crystal structure analysis at the interface of two subunits including Glu238, but it remains unknown whether this site is functionally involved in the activation of mGluR1α by Gd 3+ or not. We analyzed the ligand sensitivity of the Glu238Gln mutant, and observed that the sensitivity to extracellular Gd 3+ was completely lost, while the sensitivity to glutamate and Ca 2+ was not affected. We also observed that the presence of Gd 3+ increased the sensitivity of mGluR1α to glutamate, and that this effect was again lost by Glu238Gln mutation. These results suggest that the binding of Gd 3+ or a related endogenous substance to this site, alone or in cooperation with glutamate binding at a distant site, leads mGluR1α to activation.

Does binding of Gd‐DTPA to myocardial tissue contribute to late enhancement in a model of acute myocardial infarction?

The long-lasting signal enhancement by Gd-DTPA in areas of myocardial infarction has been conventionally explained by low perfusion and an enhanced Gd distribution volume. To test whether binding of Gd to myocardial constituents is an additional factor contributing to this effect, Gd-DTPA was allowed to equilibrate between homogenized porcine myocardial tissue and physiological saline. The relaxation rate (1/T(1)) of homogenate samples (n = 61) increased in proportion (r(2) = 0.98) to the Gd concentration (0.025-0.5 mM) of the surrounding medium, with no evidence for augmented uptake. The diffusion-limited uptake was only slightly more rapid than the subsequent Gd-release. The amount of Gd released was in line with all of the Gd-DTPA in the homogenate participating in water proton relaxation. The data from this acute myocardial infarction model do not support the notion that Gd-DTPA binding in the early stages of myocardial damage contributes to delayed enhancement.

Structure-based analysis of the herpes simplex virus glycoprotein D binding site present on herpesvirus entry mediator HveA (HVEM).

Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular beta-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.

Rabies Virus Glycoprotein (RVG) Is a Trimeric Ligand for the N-terminal Cysteine-rich Domain of the Mammalian p75 Neurotrophin Receptor

Rabies virus glycoprotein (RVG) is a trimeric and surface-exposed viral coat protein that has been shown to interact with the murine p75 neurotrophin receptor. We have investigated binding of RVG to p75 and describe several features that distinguish the p75-RVG interaction from conventional neurotrophin binding to p75. RVG binds mammalian but not avian p75 and does not bind to any of the Trk neurotrophin receptors. The mammalian p75 specificity of RVG binding may partly explain the phyletic specificity of rabies infection. Radioiodinated nerve growth factor (NGF) and RVG both bind to rat p75 but do not compete with each other's binding site. Although neurotrophins bind to the second and third cysteine-rich domains (CRD) of p75, RVG specifically interacts with high affinity (K(d) 30-35 pm) with the first CRD (CRD1). Substitution of Gln(33) in p75-CRD1 by Glu completely abolishes RVG binding. Our data therefore firmly establish RVG as a trimeric high affinity ligand for a non-neurotrophin binding site on p75. Interestingly, the CRD1 in another TNF/NGF family receptor was recently shown to be involved in the binding of the herpes virus glycoprotein gD, suggesting that the CRD1 of TNF/NGF family members may be a widely used binding domain for viral glycoproteins.

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.

Amino acid substitutions in the V domain of nectin-1 (HveC) that impair entry activity for herpes simplex virus types 1 and 2 but not for Pseudorabies virus or bovine herpesvirus 1.

The entry of herpes simplex virus (HSV) into cells requires the interaction of viral glycoprotein D (gD) with a cellular gD receptor to trigger the fusion of viral and cellular membranes. Nectin-1, a member of the immunoglobulin superfamily, can serve as a gD receptor for HSV types 1 and 2 (HSV-1 and HSV-2, respectively) as well as for the animal herpesviruses porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1). The HSV-1 gD binding domain of nectin-1 is hypothesized to overlap amino acids 64 to 104 of the N-terminal variable domain-like immunoglobulin domain. Moreover, the HSV-1 and PRV gDs compete for binding to nectin-1. Here we report that two amino acids within this region, at positions 77 and 85, are critical for HSV-1 and HSV-2 entry but not for the entry of PRV or BHV-1. Replacement of either amino acid 77 or amino acid 85 reduced HSV-1 and HSV-2 gD binding but had a lesser effect on HSV entry activity, suggesting that weak interactions between gD and nectin-1 are sufficient to trigger the mechanism of HSV entry. Substitution of both amino acid 77 and amino acid 85 in nectin-1 significantly impaired entry activity for HSV-1 and HSV-2 and eliminated binding to soluble forms of HSV-1 and HSV-2 gDs but did not impair the entry of PRV and BHV-1. Thus, amino acids 77 and 85 of nectin-1 form part of the interface with HSV gD or influence the conformation of that interface. Moreover, the binding sites for HSV and PRV or BHV-1 gDs on nectin-1 may overlap but are not identical.

Substitution in the Murine Nectin1 Receptor of a Single Conserved Amino Acid at a Position Distal from the Herpes Simplex Virus gD Binding Site Confers High-Affinity Binding to gD

By analogy with its human nectin1 counterpart, murine nectin1 serves as a cellular receptor for the entry of herpes simplex virus (HSV) into murine cells. HSV entry mediated by either receptor is dependent on the viral glycoprotein D (gD). Whereas human nectin1 binds gD at high affinity and in a saturable manner, murine nectin1 binds gD in a barely detectable fashion, depending on the sensitivity of the assay. The immunoglobulin type V domain of murine nectin differs from its human counterpart in 11 amino acids. To identify the key residues responsible for the high-affinity binding of gD to human nectin1, we replaced each of the 11 divergent amino acids with the human counterparts singly or in groups in an incremental manner. Replacement in murine nectin1 of six amino acids that lie within the gD binding region of human nectin1 (previously mapped to residues 64 to 94, likely the CC'C" surface) increased the gD binding activity to a limited extent. In contrast, the single P138L substitution, which lies distal from the gD binding site, markedly increased gD binding. This substitution, when coupled with downstream substitutions, exerted the greatest effect. Three-dimensional modeling of the nectin1 V domain suggested that P138 in murine nectin1 might decrease the stability of the V domain by reducing the size of beta-strand G. The results support the notion that the overall structure of V nectin1 plays a pivotal role in its ability to bind HSV gD.

Crystallization and preliminary diffraction studies of the ectodomain of the envelope glycoprotein D from herpes simplex virus 1 alone and in complex with the ectodomain of the human receptor HveA.

Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) cell entry. The gD ectodomain consists of 316 residues and has no sequence homology to any other proteins of known structure. Two fragments of the HSV-1 gD ectodomain (gD(22-260): residues 22-260 and gD(285): residues 1-285) have been crystallized in two crystal forms. The complex between gD(285) and the ectodomain of HveA, a gD cellular receptor member of the tumor necrosis factor (TNFR) superfamily, has also been crystallized. Moreover, insect-cell-expressed selenomethionine-substituted gD(285) has been purified and crystallized alone and in complex with HveA.

Effects of herpes simplex virus on structure and function of nectin-1/HveC.

Herpes simplex virus (HSV) entry requires the interaction between the envelope glycoprotein D (gD) and a cellular receptor such as nectin-1 (also named herpesvirus entry mediator C [HveC]) or HveA/HVEM. Nectin-1 is a cell adhesion molecule found at adherens junctions associated with the cytoplasmic actin-binding protein afadin. Nectin-1 can act as its own ligand in a homotypic interaction to bridge cells together. We used a cell aggregation assay to map an adhesive functional site on nectin-1 and identify the effects of gD binding and HSV early infection on nectin-1 function. Soluble forms of nectin-1 and anti-nectin-1 monoclonal antibodies were used to map a functional adhesive site within the first immunoglobulin-like domain (V domain) of nectin-1. This domain also contains the gD-binding site, which appeared to overlap the adhesive site. Thus, soluble forms of gD were able to prevent nectin-1-mediated cell aggregation and to disrupt cell clumps in an affinity-dependent manner. HSV also prevented nectin-1-mediated cell aggregation by occupying the receptor. Early in infection, nectin-1 was not downregulated from the cell surface. Rather, detection of nectin-1 changed gradually over a 30-min period of infection, as reflected by a decrease in the CK41 epitope and an increase in the CK35 epitope. The level of detection of virion gD on the cell surface increased within 5 min of infection in a receptor-dependent manner. These observations suggest that cell surface nectin-1 and gD may undergo conformational changes during HSV entry as part of an evolving interaction between the viral envelope and the cell plasma membrane.

Kinetic Analysis of Glycoprotein C of Herpes Simplex Virus Types 1 and 2 Binding to Heparin, Heparan Sulfate, and Complement Component C3b

Glycoprotein C (gC) from herpes simplex virus (HSV) facilitates virus entry by attaching the virion to host cell-surface heparan sulfate (HS). Although gC from HSV-1 (gC1) and from HSV-2 (gC2) bind to heparin, gC2 is believed to play a less significant role than gC1 in attachment of virus to cells. This attachment step is followed by the binding of gD to one of several cellular receptors. gC also plays an important role in immune evasion by binding to the C3b fragment of the third component of the host complement system. Yet, although both gC1 and gC2 protect HSV against complement-mediated neutralization, only gC on HSV-1-infected cells acts as a receptor for C3b. We used optical biosensor technology to quantitate the affinities (KD) and the stabilities (koff) between both serotypes of gC with heparin, HS, and C3b to address three questions concerning gC interactions. First, can differences in affinity or stability account for differences between the contributions of HSV-1 and HSV-2 gC in attachment? Our data show that the gC2–HS complex is highly unstable (koff = 0.2 s−1) compared to the gC1–HS complex (koff = 0.003 s−1), suggesting why gC2 may not play an important role in attachment of virus to cells as does gC1. Second, does gC2 have a lower affinity for C3b than does gC1, thereby explaining the lack of C3b-receptor activity on HSV-2 infected cells? Surprisingly, gC2 had a 10-fold higher affinity for C3b compared to gC1, so this functional difference in serotypes cannot be accounted for by affinity. Third, do differences in gC–HS and gD-receptor affinities support a model of HSV entry in which the gC–HS interaction is of lower affinity than the gD-receptor interaction? Our biosensor results indicate that gC has a higher affinity for HS than gD does for cellular receptors HveA (HVEM) and HveC (nectin-1).

Gastrointestinal enhancement of MRI with melanin derived from tea leaves ( Thea sinensis Linn.)

Melanin was extracted from tea leaves ( Thea sinensis Linn.) for the first time. Characterization of melanin proved similarity of the original compound to standard melanin. The Langmuir adsorption isotherms for gadolinium (Gd) binding were obtained using melanin. Melanin–Gd preparation demonstrated low acute toxicity. LD 50 for this preparation was in a range of 1250–1500 mg/kg in mice. Magnetic Resonance Imaging (MRI) properties of melanin itself and melanin–Gd complexes have been estimated. Gd free melanin fractions possess slighter relaxivity compared with its complexes. The relaxivity of lower molecular weight fraction was two times higher than relaxivity of Gd(DTPA) standard. Postcontrast images demonstrate that oral administration of melanin complexes in concentration 0.1 mM provides essential enhancement to longitudinal relaxation times (T 1)-weighted spin echo image. The required contrast and delineation of the stomach wall demonstrated uniform enhancement of MRI with proposed melanin complex.

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.

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.

Dipole Potentials Indicate Restructuring of the Membrane Interface Induced by Gadolinium and Beryllium Ions

The dipole component of the membrane boundary potential, ϕ d, is an integral parameter that may report on the conformational state of the lipid headgroups and their hydration. In this work, we describe an experimental approach to measurements of the dipole potential changes, Δ ϕ d, and apply it in studies of Be 2+ and Gd 3+ interactions with membranes composed of phosphatidylserine (PS), phosphatidylcholine (PC), and their mixtures. Δ ϕ d is determined as the difference between the changes of the total boundary potential, ϕ b, measured by the IFC method in planar lipid membranes and the surface potential, ϕ s, determined from the electrophoretic mobility of liposomes. The Gouy–Chapman–Stern formalism, combined with the condition of mass balance, well describes the ion equilibria for these high-affinity cations. For the adsorption of Be 2+ and Gd 3+ to PC membranes, and of Mg 2+ to PS membranes, the values of Δ ϕ b and Δ ϕ s are the same, indicative of no change of ϕ d. Binding of Gd 3+ to PS-containing membranes induces changes of ϕ d of opposite signs depending on the density of ionized PS headgroups in the bilayer. At low density, the induced Δ ϕ d is negative (−30 mV), consistent with the effect of dehydration of the surface. At maximal density (pure PS, neutral pH), adsorption of Be 2+ or Gd 3+ induces an increase of ϕ d of 35 or 140 mV, respectively. The onset of the strong positive dipole effect on PS membranes with Gd 3+ is observed near the zero charge point and correlates with a six-fold increase of membrane tension. The observed phenomena may reflect concerted reorientation of dipole moments of PS headgroups as a result of ion adsorption and lipid condensation. Their possible implications to in-vivo effects of these high-affinity ions are discussed.

Comparison of Murine and Human Nectin1 Binding to Herpes Simplex Virus Glycoprotein D (gD) Reveals a Weak Interaction of Murine Nectin1 to gD and a gD-Dependent Pathway of Entry

The murine nectin1α (mNectin1α), a homolog of human nectin1α (hNectin1α, or PRR1, HveC), mediates the entry of herpes simplex virus (HSV) into cells. Previously, we reported that the binding of hNectin1 to HSV glycoprotein D (gD) was readily detectable, whereas the binding of mNectin1 to gD was not detectable, thus raising the question whether mNectin1 mediates a gD-dependent or a gD-independent pathway of entry. Here we report comparative binding studies of murine- and human-nectin1α to virions and to gD. The assays consistently showed either a very weak binding or no detectable binding of murine nectin1α to gD. They included (i) binding of soluble mNectin1-Fc or hNectin1-Fc to virions and competition of the binding by soluble gD(Δ290–299t) and by monoclonal antibodies to gD; (ii) pull-down experiments of wt gD from lysates of infected cells; and (iii) ELISA binding of soluble gD(Δ290–299t) to cells expressing mNectin1 or hNectin1. In contrast to the binding studies, the entry studies readily showed that entry mediated by mNectin1 was dependent on gD. Thus, a gDnull (gD−/−) mutant virus was unable to enter mNectin1-expressing cells, and entry of wild-type virus was inhibited by antibodies to gD or soluble gD at similar concentrations. We infer that gD represents a weak ligand in the interaction between mNectin1 and virions, whereas it represents a strong and the major ligand for hNectin1. Yet gD is required in HSV-1 entry mediated by mNectin1α. We conclude that a high-affinity binding of the receptor to gD is not a requirement in the gD-dependent pathway of HSV entry to cells.

Porcine HveC, a Member of the Highly Conserved HveC/Nectin 1 Family, Is a Functional Alphaherpesvirus Receptor

Human herpesvirus entry mediator C (HveC) is an alphaherpesvirus receptor which binds to virion glycoprotein D (gD). We identified porcine HveC and studied its interaction with pseudorabies virus (PrV) and herpes simplex virus type 1 (HSV-1) gD. Porcine and human HveC have 96% amino acid identity and HveC from African green monkey, mouse, hamster, and cow are similarly conserved. Porcine HveC mediates entry of HSV-1, HSV-2, PrV, and bovine herpesvirus type 1. Truncated soluble forms of HSV-1 and PrV gD bind competitively to porcine HveC. Biosensor analysis shows that PrV gD binds with a 10-fold higher affinity than HSV-1 gD. Monoclonal antibodies against human HveC recognize the porcine homologue and can block gD binding and entry of HSV-1 and PrV. Porcine HveC is functionally indistinguishable from human HveC. Our results are consistent with the suggestion that HveC is a pan-alphaherpesvirus receptor that interacts with a conserved structural domain of gD.

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

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.

Localization of the gD-binding region of the human herpes simplex virus receptor, HveA.

During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry mediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that gD binds the ectodomain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To localize the gD-binding domain of HveA, we expressed three additional soluble forms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA(77-120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme-linked immunosorbent assay studies showed that gD bound to HveA(120t) and HveA(200t) with the same affinity. However, gD did not bind to HveA(76t) or HveA(77-120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77-120t), blocked herpes simplex virus (HSV) entry into CHO cells expressing HveA. We also generated six monoclonal antibodies (MAbs) against HveA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. None of these MAbs blocked the binding of gD to HveA. In contrast, MAb CW3 recognized a discontinuous epitope within the first CRP of HveA, blocked the binding of gD to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA contains at least a portion of the gD binding site. The inability of gD to bind HveA(76t) suggests that additional amino acid residues of the gD binding site may reside within the second CRP.

Localization of a binding site for herpes simplex virus glycoprotein D on herpesvirus entry mediator C by using antireceptor monoclonal antibodies.

The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.

The three HveA receptor ligands, gD, LT-α and LIGHT bind to distinct sites on HveA

The herpes virus entry mediator A (HveA), a member of the tumor necrosis factor receptor (TNFR) superfamily, interacts with three different protein ligands; lymphotoxin-α (LT-α) and LIGHT (LIGHT stands for lymphotoxin homolog, which exhibits inducible expression and competes with HSV glycoprotein D for HveA and is expressed on T-lymphocytes) from the host and the herpes simplex virus (HSV) surface glycoprotein gD. It has been reported that the gD binding site on HveA is located within the receptor's two N-terminal CRP domains, and that gD and LIGHT compete for their binding to HveA. However, whether these ligands interact with the same or different sites on the receptor is unclear. We analyzed and compared the sites of interaction between HveA and its TNF ligands, by using two recombinant forms of the receptor, comprising the full-receptor ectodomain (HveA (200t)) and its two first CRP domains (HveA (120t)), as well as several monoclonal antibodies recognizing HveA. Two HveA peptide ligands (BP-1 and BP-2) that differentially inhibit binding of soluble gD and LT-α to the receptor were also used to demonstrate that gD, LIGHT and LT-α bind to distinct sites on the receptor. Our results suggest that binding of a ligand to HveA may alter the conformation of this receptor, thereby affecting its interaction with its other ligands.