Herpes Simplex Virus Glycoprotein Research Articles

LIGHT sensitizes IFN-gamma-mediated apoptosis of HT-29 human carcinoma cells through both death receptor and mitochondria pathways.

LIGHT [homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM/TR2)] is a new member of TNF superfamily. The HT-29 colon cancer cell line is the most sensitive to LIGHT-induced, IFNg-mediated apoptosis among the cell lines we have examined so far. Besides downregulation of Bcl-XL, upregulation of Bak, and activation of both PARP [poly (ADP-ribose) polymerase] and DFF45 (DNA fragmentation factor), LIGHT-induced, IFNg-mediated apoptosis of HT-29 cells involves extensive caspase activation. Caspase-8 and caspase-9 activation, as shown by their cleavages appeared as early as 24 h after treatment, whereas caspase-3 and caspase-7 activation, as shown by their cleavages occurred after 72 h of LIGHT treatment. Caspase-3 inhibitor Z-DEVD-FMK (benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone) and a broad range caspase inhibitor Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) were able to block LIGHT-induced, IFNg-mediated apoptosis of HT-29 cells. The activity of caspase-3, which is one of the major executioner caspases, was found to be inhibited by both Z-DEVD-MFK and Z-VAD-FMK. These results suggest that LIGHT-induced, IFNg-mediated apoptosis of HT-29 cells is caspase-dependent, and LIGHT signaling is mediated through both death receptor and mitochondria pathways.

Long-term persistence of HIV-1 vaccine-induced CD4+CD45RA-CD62L-CCR7- memory T-helper cells.

To determine in chimpanzees if candidate HIV-1 subunit protein vaccines were capable of eliciting long-lasting T-cell memory responses in the absence of viral infection, and to determine the specific characteristics of these responses. A longitudinal study of cell-mediated immune responses induced in three chimpanzees following immunization with subunit envelope glycoproteins of either HIV-1 or herpes simplex virus (HSV)-2. Following these pre-clinical observations, four human volunteers who had been immunized 7 years previously with the same HIV-1 vaccine candidate donated blood for assessment of immune responses. Responses were monitored by protein and peptide based ELISpot assays, lymphocyte proliferation, and intracellular cytokine staining. Humoral responses were assessed by enzyme-linked immunosorbent assay and virus neutralization assays. Although antigen (Ag)-specific CD4 T-cell responses persisted for at least 5 years in chimpanzees, CD8 T-cell responses were discordant and declined within 2 years. Detailed cellular analyses revealed that strong Th1 in addition to Th2 type responses were induced by AS2/gp120 and persisted, whereas CD8 T-cell memory declined in peripheral blood. The specificity of both Th and cytotoxic T-lymphocyte responses revealed that the majority of responses were directed to conserved epitopes. The remarkable persistence of Ag-specific CD4 T-cell memory was characterized as a population of the CD45RA-CD62L-CCR7- "effector phenotype" producing the cytokines IFNgamma, IL-2 and IL-4 upon epitope-specific recognition. Importantly, results in chimpanzees were confirmed in peripheral blood of one of four human volunteers studied more than 7 years after immunization. These studies demonstrate that epitope-specific Th1 and Th2 cytokine-dependent Th responses can be induced and maintained for longer than 5 years by immunization with subunit proteins of HIV-1.

Prevention of genital herpes in a guinea pig model using a glycoprotein D-specific single chain antibody as a microbicide

BackgroundGenital herpes (GH) is a recurrent sexually transmitted infection (STI) that causes significant morbidity and is also the major source of herpes simplex virus (HSV) in cases of neonatal herpes. Vaccination is a current goal which has had limited success so far in preventing GH and microbicides offer an attractive alternative. Treatment of primary disease cannot prevent establishment of latent infections and thus, cannot prevent subsequent recurrent disease. Recently, many of the molecular events leading to entry of HSV into cells have been elucidated, resulting in the description of a number of herpesvirus entry mediators (HVEMs) that interact with HSV glycoprotein D (gD) on the surface of virions. Described here is a strategy for interrupting the spread of HSV based on interfering with these interactions. The hypothesis addressed in the current report was that single chain antibody variable fragments (scFv) that interrupt associations between gD and HVEMs would not only prevent infection in vitro but could also be used as microbicides to interfere with acquisition GH.Results and ConclusionsHere we show that a scFv derived from a particular hybridoma, DL11, not only inhibits infection in vitro but also prevents development of GH in a guinea pig model when applied intravaginally in an inert vehicle. Comparison of different anti-gD single chain antibodies supported the hypothesis that the activity of DL11-scFv is based on its ability to disrupt the associations between gD and the two major receptors for HSV, nectin-1 and HveA. Further, the results predict that bacterial expression of active single chain antibodies can be optimized to manufacture inexpensively a useful microbicidal product active against HSV.

Assessment of a cellular vaccination approach consisting of crawling dendritic cells (CDCs) transduced with HSV-1-Deltapac vectors.

Crawling dendritic cells (CDCs) and herpes simplex virus-1 (HSV-1) amplicon vectors were utilized in this study: (1) to evaluate whether CDCs can be transduced by HSV-1 amplicon vectors; (2) to assess the effects of HSV-1 infections on structure and functions of CDCs; (3) to assess the capabilities of the transduced CDC to express, process, and present the transgene products; and (4) to induce in vitro and in vivo priming of T cells and B cells. CDC supported amplicon-mediated transgene expression while retaining the ability to perform mixed lymphocyte reaction (MLR) and priming of naive T cells. Then it was tested whether transduced CDC were able to initiate immunity against either the amplicon particle and/or the product encoded by the delivered transgene by injecting groups of mice with transduced CDCs expressing GFP or LacZ. Spleen cells of these mice were stimulated by co-incubation with cells expressing: (1) either one of the transgenes (GFP or LacZ), (2) peptides of beta-gal, or (3) peptides of HSV-1 glycoprotein B (gB). Interestingly, no significant cytotoxic T lymphocyte (CTL) activity against the transgenes or against gB was observed. In contrast, mice developed high levels of antibodies against gB and LacZ.Mainly, the findings that CDCs not only express amplicon-delivered transgene, but were able to induce MLR and priming of naïve T cells against the transduced antigen, open up unexpected possibilities and the likelihood to use CDCs as a vehicle for cellular immunization against any transduced antigens. However, these results indicate that HSV-1 amplicon-transduced CDCs induce effective priming and a humoral response, but no strong cell-mediated immune response.

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.

Blocking immune evasion as a novel approach for prevention and treatment of herpes simplex virus infection.

Many microorganisms encode immune evasion molecules to escape host defenses. Herpes simplex virus type 1 glycoprotein gC is an immunoevasin that inhibits complement activation by binding complement C3b. gC is expressed on the virus envelope and infected cell surface, which makes gC potentially accessible to blocking antibodies. Mice passively immunized with gC monoclonal antibodies prior to infection were protected against herpes simplex virus challenge only if the gC antibodies blocked C3b binding. Mice treated 1 or 2 days postinfection with gC monoclonal antibodies that block C3b binding had less severe disease than control mice treated with nonimmune immunoglobulin G (IgG). Mice immunized with gC protein produced antibodies that blocked C3b binding to gC. Immunized mice were significantly protected against challenge by wild-type virus, but not against a gC mutant virus lacking the C3b binding domain, suggesting that protection was mediated by antibodies that target the gC immune evasion domain. IgG and complement from subjects immunized with an experimental herpes simplex virus glycoprotein gD vaccine neutralized far more mutant virus defective in immune evasion than wild-type virus, supporting the importance of immune evasion molecules in reducing vaccine potency. These results suggest that it is possible to block immune evasion domains on herpes simplex virus and that this approach has therapeutic potential and may enhance vaccine efficacy.

The cytoplasmic tail of Herpes simplex virus glycoprotein H binds to the tegument protein VP16 in vitro and in vivo

During Herpes simplex virus envelopment, capsids, tegument polypeptides, and membrane proteins assemble at the site of budding and a cellular lipid bilayer becomes refashioned into a spherical envelope. Though the molecular interactions driving these events are poorly understood, several lines of evidence suggest that associations between envelope protein cytoplasmic tails and tegument polypeptides may play important roles. Consistent with this hypothesis, we show here that a fusion of the cytoplasmic tail of gH with Glutathione- S-Transferase binds to VP16 in a temperature-dependent manner. VP16 prepared by in vitro translation behaves in a similar fashion, demonstrating that the interaction is not dependent on other viral polypeptides. Mutational analysis of the gH tail has also enabled us to identify amino acid residues critical for VP16 binding in vitro. A fusion protein in which the gH tail is fused to the carboxy-terminus of GFP coimmunoprecipitates with VP16 in infected cells, indicating that VP16 can interact with the gH tail in vivo.

Chimeric Dr fimbriae with a herpes simplex virus type 1 epitope as a model for a recombinant vaccine.

The potential of the major structural protein DraE of Escherichia coli Dr fimbriae has been used to display an 11-amino-acid peptide of glycoprotein D derived from herpes simplex virus (HSV) type 1. The heterologous sequence mimicking an epitope from glycoprotein D was inserted in one copy into the draE gene in place of a predicted 11-amino-acid sequence in the N-terminal region of surface-exposed domain 2 within the conserved disulfide loop (from Cys21 to Cys53). The inserted epitope was displayed on the surface of the chimeric DraE protein as evidenced by immunofluorescence and was recognized by monoclonal antibodies to the target HSV glycoprotein D antigen. Conversely, immunization of rabbits with purified chimeric Dr-HSV fimbriae resulted in a serum that specifically recognized the 11-amino-acid epitope of HSV glycoprotein D, indicating the utility of the strategy employed.

Protection by Herpes Simplex Virus Glycoprotein D against Fas-mediated Apoptosis: ROLE OF NUCLEAR FACTOR κB

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-kappaB was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-kappaBalpha, indicating that NF-kappaB activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-kappaB-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity.

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.

Herpes simplex virus glycoproteins gD and gE/gI serve essential but redundant functions during acquisition of the virion envelope in the cytoplasm.

The late stages of assembly of herpes simplex virus (HSV) and other herpesviruses are not well understood. Acquisition of the final virion envelope apparently involves interactions between viral nucleocapsids coated with tegument proteins and the cytoplasmic domains of membrane glycoproteins. This promotes budding of virus particles into cytoplasmic vesicles derived from the trans-Golgi network or endosomes. The identities of viral membrane glycoproteins and tegument proteins involved in these processes are not well known. Here, we report that HSV mutants lacking two viral glycoproteins, gD and gE, accumulated large numbers of unenveloped nucleocapsids in the cytoplasm. These aggregated capsids were immersed in an electron-dense layer that appeared to be tegument. Few or no enveloped virions were observed. More subtle defects were observed with an HSV unable to express gD and gI. A triple mutant lacking gD, gE, and gI exhibited more severe defects in envelopment. We concluded that HSV gD and the gE/gI heterodimeric complex act in a redundant fashion to anchor the virion envelope onto tegument-coated capsids. In the absence of either one of these HSV glycoproteins, envelopment proceeds; however, without both gD and gE, or gE/gI, there is profound inhibition of cytoplasmic envelopment.

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.

Herpes simplex virus glycoprotein K, but not its syncytial allele, inhibits cell-cell fusion mediated by the four fusogenic glycoproteins, gD, gB, gH, and gL.

A Myc epitope was inserted at residue 283 of herpes simplex virus type 1 (HSV-1) glycoprotein K (gK), a position previously shown not to interfere with gK activity. The Myc-tagged gK localized predominantly to the endoplasmic reticulum, both in uninfected and in HSV-infected cells. gK, coexpressed with the four HSV fusogenic glycoproteins, gD, gB, gH, and gL, inhibited cell-cell fusion. The effect was partially dose dependent and was observed both in baby hamster kidney (BHK) and in Vero cells, indicating that the antifusion activity of gK may be cell line independent. The antifusion activity of gK did not require viral proteins other than the four fusogenic glycoproteins. A syncytial (syn) allele of gK (syn-gK) carrying the A40V substitution present in HSV-1(MP) did not block fusion to the extent seen with the wild-type (wt) gK, indicating that the syn mutation ablated, at least in part, the antifusogenic activity of wt gK. We conclude that gK is part of the mechanism whereby HSV negatively regulates its own fusion activity. Its effect accounts for the notion that cells infected with wt HSV do not fuse with adjacent, uninfected cells into multinucleated giant cells or syncytia. gK may also function to preclude fusion between virion envelope and the virion-encasing vesicles during virus transport to the extracellular compartment, thus preventing nucleocapsid de-envelopment in the cytoplasm.

Optical biosensor analysis in studying herpes simplex virus glycoprotein D binding to target nectin1 receptor

Studies on molecular interactions between cellular receptors of herpes simplex virus (HSV) and the viral glycoproteins showing receptor-binding activity are of great relevance for understanding the molecular basis of virus entry. Information on such interactions further provides the basis for a rational design of antiviral drugs. A variety of biochemical and biophysical methodologies are used for determining the binding parameters of interacting biomolecules. Most of them require relatively high amounts of the analyzed compounds, or the use of labeled target molecules. Here, we report the study of the binding of two recombinant forms of HSV glycoprotein D, gD(Δ290-299t) and gD(305t), and a recombinant form of the human cellular receptor for HSV, nectin1-Fc, by using an optical biosensor (IAsys Plus, Affinity Sensors, UK). This device detects and quantifies the changes in refractive index in the vicinity of the surface of sensor chips to which ligands are immobilized. The changes in the refractive index are proportional to the change in the absorbed mass, thus the analysis allows the monitoring of the interaction process in real-time and the determination of the binding parameters. HSV cellular receptor has been immobilized on the surface of the biosensor cuvette, bearing a carboxymethyl dextran layer. The immobilized receptor cuvette was then used for the binding experiments of the two glycoproteins. A significant difference in their dissociation constants was determined, showing for the gD(Δ290-299t) protein a much higher affinity ( K D, 2.8×10 −7 M) with respect to gD(305t) ( K D, 2.8×10 −6 M). The active ligand concentration decreased on time, however the binding properties of the immobilized receptor were maintained over 5 weeks.

The Role of Apoptosis Signal-regulating Kinase 1 in Lymphotoxin-β Receptor-mediated Cell Death

LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes) is a member of the tumor necrosis factor superfamily that can interact with lymphotoxin-beta receptor (LTbetaR), herpes virus entry mediator, and decoy receptor (DcR3). In our previous study, we showed that LIGHT is able to induce cell death via the non-death domain containing receptor LTbetaR to activate both caspase-dependent and caspase-independent pathway. In this study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar binding specificity as wild type LIGHT to LTbetaR, but lose the ability to interact with herpes virus entry mediator. By using both LIGHT-R228E and agonistic anti-LTbetaR monoclonal antibody, we found that signaling triggered by LTbetaR alone is sufficient to activate both caspase-dependent and caspase-independent pathways. Cross-linking of LTbetaR is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of caspase-3 activation, and kinase-inactive ASK1-KE mutant can inhibit LTbetaR-mediated cell death. This suggests that ASK1 is one of the factors involved in the caspase-independent pathway of LTbetaR-induced cell death.

Enhancement of DNA vaccine potency against herpes simplex virus 1 by co-administration of an interleukin-18 expression plasmid as a genetic adjuvant.

In this study, the immune-modulatory and vaccine effects of using an interleukin (IL)-18 expression plasmid as a genetic adjuvant to enhance DNA vaccine-induced immune responses were investigated in a mouse herpes simplex virus 1 (HSV-1) challenge model. BALB/c mice were immunized by three intramuscular inoculations of HSV-1 glycoprotein D (gD) DNA vaccine alone or in combination with a plasmid expressing mature IL-18 peptide. Both the serum IgG2a/IgG1 ratio and T helper 1-type (Th1) cytokines [IL-2 and interferon (IFN)-gamma] were increased significantly by the co-injection of the IL-18 plasmid compared with the injection of gD DNA alone. However, the production of IL-10 was inhibited by IL-18 plasmid co-injection. Furthermore, IL-18 plasmid co-injection efficiently enhanced antigen-specific lymphocyte proliferation and the delayed-type hypersensitivity response. When mice were challenged with HSV-1 at the cornea, co-injection of IL-18 plasmid with gD DNA vaccine showed significantly better protection, manifested as lower corneal lesion scores and faster recovery. These experiments indicate that co-injection of an IL-18 plasmid with gD DNA vaccine efficiently induces Th1-dominant immune responses and improves the protective effect against HSV-1 infection.

Light stimulates IFNγ-Mediated intercellular adhesion molecule-1 upregulation of cancer cells

Intercellular adhesion molecule-1 (ICAM-1) works as one of the ligands for activating the killing activity of natural killer (NK) cells and cancer specific cytotoxic T lymphocytes (CTL). Expression of ICAM-1 enhances lymphocyte adhesion to the cancer cells in vivo. Cancer cell lines express significantly lower level of ICAM-1 than that of normal epithelium or benign cells. Overexpression of LIGHT (LIGHT: homologous to lymphotoxins, indicating inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator [HVEM/TR2]) in MDA-MB-231 human breast cancer cells was observed to suppress tumor growth in vivo. In order to elucidate the mechanisms how LIGHT overexpression could trigger tumor suppression, the expression level of a panel of cell surface makers CD54, CD56, CD95, and CD119 was investigated in a group of cancer cells. Flow cytometry analysis results demonstrate that LIGHT gene expression in cancer cells can greatly increase ICAM-1 expression level, IFNγ alone can stimulate cancer cells to express ICAM-1, which can be highly augmented by LIGHT in a dose-dependent manner. This upregulation of ICAM-1 expression is not only at ICAM-1 protein trafficking level on cell surface as demonstrated by flow cytometry analysis, but also at ICAM-1 total protein level as confirmed by Western blot. There is no difference of expression level among these cancer cell lines for the other three cell surface markers: CD56, CD95 (Fas), and CD119. It was confirmed that LIGHT enhancement upregulation of ICAM-1 expression is at least STAT1 and JAK1 dependent by using STAT1-deficient U3A and JAK1-deficient E2A4 cells. These findings suggest that LIGHT-induced inhibition of tumor growth is highly correlated with its upregulation of ICAM-1 expression.

Herpes Simplex Virus gE/gI Expressed in Epithelial Cells Interferes with Cell-to-Cell Spread

The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.

Differential Expression of LIGHT and Its Receptors in Human Placental Villi and Amniochorion Membranes

mRNA encoding LIGHT (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), a member of the tumor necrosis factor superfamily of ligands, as well as mRNAs encoding LIGHT receptors [HVEM, LTbetaR, and TR6 (DcR3)] are present in placentas and cytotrophoblast cells at term. To establish translation of these messages and determine directions for functional studies, term placentas, amniochorion membranes, and purified cytotrophoblast cells were evaluated by immunoblotting and immunohistochemistry. Ligand and receptor proteins were identified in lysates from all three sources although the soluble receptor, TR6, was scarce in placentas and all receptors were in low abundance in cytotrophoblast cells. These results were confirmed and cell type-specific expression was documented by immunohistochemistry. Ligand and receptor proteins were differentially expressed according to cell type. For example, HVEM was identified on syncytiotrophoblast but not in villous mesenchymal cells; amnion epithelial cells were positive for all proteins whereas chorion membrane cytotrophoblasts exhibited none. Because LIGHT is a powerful cytokine that can alter gene expression and promote apoptosis, these experiments suggest that ligand-receptor interactions may critically influence structural and functional aspects of human placentas through as yet undefined autocrine/paracrine pathways.

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.