Herpes Simplex Virus Glycoprotein D Can Bind to Poliovirus Receptor-Related Protein 1 or Herpesvirus Entry Mediator, Two Structurally Unrelated Mediators of Virus Entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.     

Stable association of herpes simplex virus with target membranes is triggered by low pH in the presence of the gD receptor, HVEM

Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37 degrees C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37 degrees C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.     

Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry.

Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection.

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.     

Examination of the Kinetics of Herpes Simplex Virus Glycoprotein D Binding to the Herpesvirus Entry Mediator, Using Surface Plasmon Resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (K_D) of 3.2 × 10⁻⁶ M and gD2(306t) had a K_D of 1.5 × 10⁻⁶ M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Δ290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (K_D = 3.3 × 10⁻⁸ M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Δ290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Δ290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Δ290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 × 10⁻⁸ M versus 3.2 × 10⁻⁶ M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Δ290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster k_on rather than to a slower k_off. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 × 10⁻⁵ M) than did gD1(306t) due to a more rapid k_off. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

The major neutralizing antigenic site on herpes simplex virus glycoprotein D overlaps a receptor-binding domain

Herpes simplex virus (HSV) entry is dependent on the interaction of virion glycoprotein D (gD) with one of several cellular receptors. We previously showed that gD binds specifically to two structurally dissimilar receptors, HveA and HveC. We have continued our studies by using (i) a panel of baculovirus-produced gD molecules with various C-terminal truncations and (ii) a series of gD mutants with nonoverlapping 3-amino-acid deletions between residues 222 and 254. Binding of the potent neutralizing monoclonal antibody (MAb) DL11 (group Ib) was unaffected in forms of gD containing residues 1 to 250 but was greatly diminished in molecules truncated at residue 240 or 234. Both receptor binding and blocking of HSV infection were also affected by these C-terminal truncations. gD-1(234t) bound weakly to both HveA and HveC as determined by enzyme-linked immunosorbent assay (ELISA) and failed to block infection. Interestingly, gD-1(240t) bound well to both receptors but blocked infection poorly, indicating that receptor binding as measured by ELISA is not the only gD function required for blocking. Optical biosensor studies showed that while gD-1(240t) bound HveC with an affinity similar to that of gD-1(306t), the rates of complex formation and dissociation were significantly faster than for gD-1(306t). Complementation analysis showed that any 3-amino-acid deletion between residues 222 and 251 of gD resulted in a nonfunctional protein. Among this set of proteins, three had lost DL11 reactivity (those with deletions between residues 222 and 230). One of these proteins (deletion 222-224) was expressed as a soluble form in the baculovirus system. This protein did not react with DL11, bound to both HveA and HveC poorly as shown by ELISA, and failed to block HSV infection. Since this protein was bound by several other MAbs that recognize discontinuous epitopes, we conclude that residues 222 to 224 are critical for gD function. We propose that the potent virus-neutralizing activity of DL11 (and other group Ib MAbs) likely reflects an overlap between its epitope and a receptor-binding domain of gD.     

The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.     

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.     

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.     

Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry

Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts.     

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.     

Targeting of antigen to the herpesvirus entry mediator augments primary adaptive immune responses

Interactions between the herpesvirus entry mediator (HVEM) and the B- and T-lymphocyte attenuator (BTLA) inhibit B and T cell activation. HVEM-BTLA interactions are blocked by herpes simplex virus (HSV) glycoprotein D (gD) through binding of its N-terminal domain to the BTLA binding site of HVEM. In this study, we inserted viral antigens into the C-terminal domain of gD and expressed these antigens with plasmid or E1-deleted (replication-defective) adenovirus vectors. Viral antigens fused to gD induced T and B cell responses to the antigen that were far more potent than those elicited by the same antigen expressed without gD. The immunopotentiating effect required binding of the gD chimeric protein to HVEM. Overall, the studies demonstrate that targeting of antigen to the BTLA binding site of HVEM augments the immunogenicity of vaccines.     

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.     

Antibody-induced conformational changes in herpes simplex virus glycoprotein gD reveal new targets for virus neutralization

As the receptor-binding protein of herpes simplex virus (HSV), gD plays an essential role in virus entry. In its native state, the last 56 amino acids of the ectodomain C terminus (C-term) occlude binding to its receptors, herpesvirus entry mediator (HVEM) and nectin-1. Although it is clear that movement of the C-term must occur to permit receptor binding, we believe that this conformational change is also a key event for triggering later steps leading to fusion. Specifically, gD mutants containing disulfide bonds that constrain the C-term are deficient in their ability to trigger fusion following receptor binding. In this report, we show that two newly made monoclonal antibodies (MAbs), MC2 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to a receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances the affinity of gD for both receptors. Several nonneutralizing MAbs (MC4, MC10, and MC14) also enhanced gD-receptor binding. While MC2 and MC5 recognized different epitopes on the core of gD, these nonneutralizing MAbs recognized the gD C-term. Both the neutralizing capacity and rate of neutralization of virus by MC2 are uniquely enhanced when MC2 is combined with MAb MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from performing a function that triggers later steps leading to fusion and that the epitope for MC2 is normally occluded by the C-term of the gD ectodomain.     

Glycoprotein D receptor-dependent, low-pH-independent endocytic entry of herpes simplex virus type 1

Two herpes simplex virus type 1 (HSV-1) entry pathways have been described: direct fusion between the virion envelope and the plasma membrane, as seen on Vero cells, and low-pH-dependent endocytosis, as seen on CHO nectin-1 and HeLa cells. In this paper, we studied HSV entry into C10 murine melanoma cells and identified a third entry pathway for this virus. During entry into C10 cells, virion envelope glycoproteins rapidly became protected from the membrane-impermeable chemical cross-linker BS3 and from proteinase K. Protection was gD receptor dependent, and the time taken to detect protected protein was proportional to the rate of virus entry. Ultrastructural examination revealed that virions attached to the surface of C10 cells were localized to membrane invaginations, whereas those on the surface of receptor-negative B78 cells were peripherally attached. Virus entry into C10 cells was energy dependent, and intracellular enveloped virions were seen within membrane-bound vesicles consistent with endocytic entry. Entry was not inhibited by bafilomycin A1 or ammonium chloride, showing that passage of the virion through a low-pH environment was not required for infection. Resistance to similar reagents should therefore not be taken as proof of HSV entry by a nonendosomal pathway. These data define a novel gD receptor-dependent acid-independent endocytic entry pathway for HSV.     

The Engineering of a Novel Ligand in gH Confers to HSV an Expanded Tropism Independent of gD Activation by Its Receptors

Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand – a single-chain antibody (scFv) to the cancer-specific HER2 receptor – expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2–gH chimera into HER2⁺ cells occurred in the absence of gD receptors, or upon deletion of key residues in gD that constitute the nectin1/HVEM binding sites. In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.     

Involvement of HVEM receptor in activation of nuclear factor kappaB by herpes simplex virus 1 glycoprotein D

The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been shown to activate nuclear factor kappaB (NF-kappaB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV-inactivated HSV-1 induced an increased NF-kappaB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose-dependent NF-kappaB activation in THP-1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF-kappaB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild-type gD on their surface induced an approximately twofold increase in NF-kappaB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose-dependent NF-kappaB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF-kappaB activation by HSV-1 gD.     

Inhibition of Herpes Simplex Virus gD and Lymphotoxin-α Binding to HveA by Peptide Antagonists

The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-alpha (LT-alpha), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-alpha, we found that BP-1 inhibited the interaction of cellular LT-alpha with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-alpha and gD, the virus-specific protein involved in HSV entry into cells.     

Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.