Trehalose enhances mitochondria deficits in human NPC1 mutant fibroblasts but disrupts mouse Purkinje cell dendritic growth ex vivo.

Lysosomes play important roles in catabolism, nutrient sensing, metabolic signaling, and homeostasis. NPC1 deficiency disrupts lysosomal function by inducing cholesterol accumulation that leads to early neurodegeneration in Niemann-Pick type C (NPC) disease. Mitochondria pathology and deficits in NPC1 deficient cells are associated with impaired lysosomal proteolysis and metabolic signaling. It is thought that activation of the transcription factor TFEB, an inducer of lysosome biogenesis, restores lysosomal-autophagy activity in lysosomal storage disorders. Here, we investigated the effect of trehalose, a TFEB activator, in the mitochondria pathology of NPC1 mutant fibroblasts in vitro and in mouse developmental Purkinje cells ex vivo. We found that in NPC1 mutant fibroblasts, serum starvation or/and trehalose treatment, both activators of TFEB, reversed mitochondria fragmentation to a more tubular mitochondrion. Trehalose treatment also decreased the accumulation of Filipin+ cholesterol in NPC1 mutant fibroblasts. However, trehalose treatment in cerebellar organotypic slices (COSCs) from wild-type and Npc1nmf164 mice caused mitochondria fragmentation and lack of dendritic growth and degeneration in developmental Purkinje cells. Our data suggest, that although trehalose successfully restores mitochondria length and decreases cholesterol accumulation in NPC1 mutant fibroblasts, in COSCs, Purkinje cells mitochondria and dendritic growth are negatively affected possibly through the overactivation of the TFEB-lysosomal-autophagy pathway.

Neutralizing antibodies against EBV gp42 show potent in vivo protection and define novel epitopes.

Epstein-Barr virus (EBV) is the first reported human oncogenic virus and infects more than 95% of the human population worldwide. EBV latent infection in B lymphocytes is essential for viral persistence. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into B cells. The C-type lectin domain (CTLD) of gp42 plays a key role in receptor binding and is the major target of neutralizing antibodies. Here, we isolated two rabbit antibodies, 1A7 and 6G7, targeting gp42 CTLD with potent neutralizing activity against B cell infection. Antibody 6G7 efficiently protects humanized mice from lethal EBV challenge and EBV-induced lymphoma. Neutralizing epitopes targeted by antibodies 1A7 and 6G7 are distinct and novel. Antibody 6G7 blocks gp42 binding to B cell surface and both 1A7 and 6G7 inhibit membrane fusion with B cells. Furthermore, 1A7- and 6G7-like antibodies in immunized sera are major contributors to B cell neutralization. This study demonstrates that anti-gp42 neutralizing antibodies are effective in inhibiting EBV infection and sheds light on the design of gp42-based vaccines and therapeutics.

Ex Vivo Infection of Human Skin Models with Herpes Simplex Virus 1: Accessibility of the Receptor Nectin-1 during Formation or Impairment of Epidermal Barriers Is Restricted by Tight Junctions.

Herpes simplex virus 1 (HSV-1) must overcome epidermal barriers to reach its receptors on keratinocytes and initiate infection in human skin. The cell-adhesion molecule nectin-1, which is expressed in human epidermis, acts as an efficient receptor for HSV-1 but is not within reach of the virus upon exposure of human skin under nonpathological conditions. Atopic dermatitis skin, however, can provide an entry portal for HSV-1 emphasizing the role of impaired barrier functions. Here, we explored how epidermal barriers impact HSV-1 invasion in human epidermis and influence the accessibility of nectin-1 for the virus. Using human epidermal equivalents, we observed a correlation of the number of infected cells with tight-junction formation, suggesting that mature tight junctions prior to formation of the stratum corneum prevent viral access to nectin-1. Consequently, impaired epidermal barriers driven by Th2-inflammatory cytokines interleukin 4 (IL-4) and IL-13 as well as the genetic predisposition of nonlesional atopic dermatitis keratinocytes correlated with enhanced infection supporting the impact of functional tight junctions for preventing infection in human epidermis. Comparable to E-cadherin, nectin-1 was distributed throughout the epidermal layers and localized just underneath the tight-junctions. While nectin-1 was evenly distributed on primary human keratinocytes in culture, the receptor was enriched at lateral surfaces of basal and suprabasal cells during differentiation. Nectin-1 showed no major redistribution in the thickened atopic dermatitis and IL-4/IL-13-treated human epidermis in which HSV-1 can invade. However, nectin-1 localization toward tight junction components changed, suggesting that defective tight-junction barriers make nectin-1 accessible for HSV-1 which enables facilitated viral penetration. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a widely distributed human pathogen which productively infects epithelia. The open question is which barriers of the highly protected epithelia must the virus overcome to reach its receptor nectin-1. Here, we used human epidermal equivalents to understand how physical barrier formation and nectin-1 distribution contribute to successful viral invasion. Inflammation-induced barrier defects led to facilitated viral penetration strengthening the role of functional tight-junctions in hindering viral access to nectin-1 that is localized just underneath tight junctions and distributed throughout all layers. We also found nectin-1 ubiquitously localized in the epidermis of atopic dermatitis and IL-4/IL-13-treated human skin implying that impaired tight-junctions in combination with a defective cornified layer allow the accessibility of nectin-1 to HSV-1. Our results support that successful invasion of HSV-1 in human skin relies on defective epidermal barriers, which not only include a dysfunctional cornified layer but also depend on impaired tight junctions.

Targeting herpesvirus entry complex and fusogen glycoproteins with prophylactic and therapeutic agents.

Herpesviruses are among the most successful viruses found in human populations. They establish lifelong latent infections, which are punctuated by recurrent reactivations. The entry process of herpesviruses into specific target cells requires a well-orchestrated teamwork involving multiple envelope glycoproteins. The conserved glycoprotein B (gB) is the membrane fusogen, of which conformational changes are induced by an entry complex (EC) consisting of at least gH and gL. Despite the high prevalence and heavy disease burdens associated with human herpesviruses (HHVs), vaccines against these pathogens are still lacking, except for varicella zoster virus (VZV). Recent advances in understanding the coordinated mechanisms of action of the key EC glycoproteins and fusogen will help to improve approaches for effective vaccine development and neutralizing antibody (nAb) screening.

Urgency and necessity of Epstein-Barr virus prophylactic vaccines.

Epstein-Barr virus (EBV), a γ-herpesvirus, is the first identified oncogenic virus, which establishes permanent infection in humans. EBV causes infectious mononucleosis and is also tightly linked to many malignant diseases. Various vaccine formulations underwent testing in different animals or in humans. However, none of them was able to prevent EBV infection and no vaccine has been approved to date. Current efforts focus on antigen selection, combination, and design to improve the efficacy of vaccines. EBV glycoproteins such as gH/gL, gp42, and gB show excellent immunogenicity in preclinical studies compared to the previously favored gp350 antigen. Combinations of multiple EBV proteins in various vaccine designs become more attractive approaches considering the complex life cycle and complicated infection mechanisms of EBV. Besides, rationally designed vaccines such as virus-like particles (VLPs) and protein scaffold-based vaccines elicited more potent immune responses than soluble antigens. In addition, humanized mice, rabbits, as well as nonhuman primates that can be infected by EBV significantly aid vaccine development. Innovative vaccine design approaches, including polymer-based nanoparticles, the development of effective adjuvants, and antibody-guided vaccine design, will further enhance the immunogenicity of vaccine candidates. In this review, we will summarize (i) the disease burden caused by EBV and the necessity of developing an EBV vaccine; (ii) previous EBV vaccine studies and available animal models; (iii) future trends of EBV vaccines, including activation of cellular immune responses, novel immunogen design, heterologous prime-boost approach, induction of mucosal immunity, application of nanoparticle delivery system, and modern adjuvant development.

A high-throughput neutralizing assay for antibodies and sera evaluation against Epstein-Barr virus.

BACKGROUND: Epstein-Barr virus (EBV) is a wide-spread human herpesvirus that is highly associated with infectious mononucleosis and several malignancies. Evaluation of EBV neutralizing antibody titers is important for serological studies, vaccine development and monoclonal antibody screening. The traditional method based on antibody inhibition of EBV transformation of B cells is very time-consuming. A more practical flow cytometry-based (FCM) approach to evaluate neutralizing titers is not amenable to achieving high-throughput evaluation of large-scale samples. A high-throughput approach is urgently needed. RESULTS: Here, we present a rapid and high-throughput method based on high content imaging system (HCIS) analysis. EBV titers determined by the HCIS-based assay were similar to those obtained by the FCM-based assay. Neutralizing titers of sera and monoclonal antibodies measured by the HCIS-based assay strongly correlated with titers measured by the FCM-based assay. HCIS assays showed a strong correlation between B cell infection neutralizing titers and the anti-gp350 IgG titers in healthy EBV carriers and monkey sera. Finally, anti-gHgL IgG titers from sera of healthy EBV carriers significantly correlated with epithelial cell infection neutralizing titers. CONCLUSIONS: This HCIS-based assay is a high-throughput assay to determine viral titers and evaluate neutralizing potentials of sera and monoclonal antibodies. This HCIS-based assay will aid the development of vaccines and therapeutic monoclonal antibody against EBV.

Dose-Dependent Outcome of EBV Infection of Humanized Mice Based on Green Raji Unit (GRU) Doses.

Humanized mouse models are used as comprehensive small-animal models of EBV infection. Previously, infectious doses of EBV used in vivo have been determined mainly on the basis of TD50 (50% transforming dose), which is a time-consuming process. Here, we determined infectious doses of Akata-EBV-GFP using green Raji units (GRUs), and characterized dose-dependent effects in humanized mice. We defined two outcomes in vivo, including an infection model and a lymphoma model, following inoculation with low or high doses of Akata-EBV-GFP, respectively. Inoculation with a low dose induced primary B cells to become lymphoblastoid cell lines in vitro, and caused latent infection in humanized mice. In contrast, a high dose of Akata-EBV-GFP resulted in primary B cells death in vitro, and fatal B cell lymphomas in vivo. Following infection with high doses, the frequency of CD19+ B cells decreased, whereas the percentage of CD8+ T cells increased in peripheral blood and the spleen. At such doses, a small part of activated CD8+ T cells was EBV-specific CD8+ T cells. Thus, GRUs quantitation of Akata-EBV-GFP is an effective way to quantify infectious doses to study pathologies, immune response, and to assess (in vivo) the neutralizing activity of antibodies raised by immunization against EBV.

Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL.

A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV.IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

A novel vaccine candidate based on chimeric virus-like particle displaying multiple conserved epitope peptides induced neutralizing antibodies against EBV infection.

Rationale: Epstein-Barr virus (EBV) is the causative pathogen for infectious mononucleosis and many kinds of malignancies including several lymphomas such as Hodgkin's lymphoma, Burkitt's lymphoma and NK/T cell lymphoma as well as carcinomas such as nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBV-GC). However, to date no available prophylactic vaccine was launched to the market for clinical use. Methods: To develop a novel vaccine candidate to prevent EBV infection and diseases, we designed chimeric virus-like particles (VLPs) based on the hepatitis B core antigen (HBc149). Various VLPs were engineered to present combinations of three peptides derived from the receptor binding domain of EBV gp350. All the chimeric virus-like particles were injected into Balb/C mice for immunogenicity evaluation. Neutralizing titer of mice sera were detected using an in vitro cell model. Results: All chimeric HBc149 proteins self-assembled into VLPs with gp350 epitopes displayed on the surface of spherical particles. Interestingly, the different orders of the three epitopes in the chimeric proteins induced different immune responses in mice. Two constructs (149-3A and 149-3B) induced high serum titer against the receptor-binding domain of gp350. Most importantly, these two VLPs elicited neutralizing antibodies in immunized mice, which efficiently blocked EBV infection in cell culture. Competition analysis showed that sera from these mice contained antibodies to a major neutralizing epitope recognized by the strong neutralizing mAb 72A1. Conclusion: Our data demonstrate that HBc149 chimeric VLPs provide a valuable platform to present EBV gp350 antigens and offer a robust basis for the development of peptide-based candidate vaccines against EBV.

Herpes Simplex Virus 1 Spread in Oligodendrocytic Cells Is Highly Dependent on MAL Proteolipid.

Myelin and lymphocyte protein (MAL) is a tetraspan integral membrane protein that resides in detergent-insoluble membrane fractions enriched in condensed membranes. MAL is expressed in oligodendrocytes, in Schwann cells, where it is essential for the stability of myelin, and at the apical membrane of epithelial cells, where it has a critical role in transport. In T lymphocytes, MAL is found at the immunological synapse and plays a crucial role in exosome secretion. However, no involvement of MAL in viral infections has been reported so far. Here, we show that herpes simplex virus 1 (HSV-1) virions travel in association with MAL-positive structures to reach the end of cellular processes, which contact uninfected oligodendrocytes. Importantly, the depletion of MAL led to a significant decrease in infection, with a drastic reduction in the number of lytic plaques in MAL-silenced cells. These results suggest a significant role for MAL in viral spread at cell contacts. The participation of MAL in the cell-to-cell spread of HSV-1 may shed light on the involvement of proteolipids in this process.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establish latent infections in neurons. HSV-1 may spread from infected to uninfected cells by two main routes: by cell-free virus or by cell-to-cell spread. In the first case, virions exit into the extracellular space and then infect another cell from the outside. In the second case, viral transmission occurs through cell-to-cell contacts via a mechanism that is still poorly understood. A third mode of spread, using extracellular vesicles, also exists. In this study, we demonstrate the important role for a myelin protein, myelin and lymphocyte protein (MAL), in the process of cell-to-cell viral spread in oligodendrocytes. We show that MAL is involved in trafficking of virions along cell processes and that MAL depletion produces a significant alteration in the viral cycle, which reduces cell-to cell spread of HSV-1.

Saliva enhances infection of gingival fibroblasts by herpes simplex virus 1.

Oral herpes is a highly prevalent infection caused by herpes simplex virus 1 (HSV-1). After an initial infection of the oral cavity, HSV-1 remains latent in sensory neurons of the trigeminal ganglia. Episodic reactivation of the virus leads to the formation of mucocutaneous lesions (cold sores), but asymptomatic reactivation accompanied by viral shedding is more frequent and allows virus spread to new hosts. HSV-1 DNA has been detected in many oral tissues. In particular, HSV-1 can be found in periodontal lesions and several studies associated its presence with more severe periodontitis pathologies. Since gingival fibroblasts may become exposed to salivary components in periodontitis lesions, we analyzed the effect of saliva on HSV-1 and -2 infection of these cells. We observed that human gingival fibroblasts can be infected by HSV-1. However, pre-treatment of these cells with saliva extracts from some but not all individuals led to an increased susceptibility to infection. Furthermore, the active saliva could expand HSV-1 tropism to cells that are normally resistant to infection due to the absence of HSV entry receptors. The active factor in saliva was partially purified and comprised high molecular weight complexes of glycoproteins that included secretory Immunoglobulin A. Interestingly, we observed a broad variation in the activity of saliva between donors suggesting that this activity is selectively present in the population. The active saliva factor, has not been isolated, but may lead to the identification of a relevant biomarker for susceptibility to oral herpes. The presence of a salivary factor that enhances HSV-1 infection may influence the risk of oral herpes and/or the severity of associated oral pathologies.

Interaction between nectin-1 and the human natural killer cell receptor CD96.

Regulation of Natural Killer (NK) cell activity is achieved by the integration of both activating and inhibitory signals acquired at the immunological synapse with potential target cells. NK cells express paired receptors from the immunoglobulin family which share common ligands from the nectin family of adhesion molecules. The activating receptor CD226 (DNAM-1) binds to nectin-2 and CD155, which are also recognized by the inhibitory receptor TIGIT. The third receptor in this family is CD96, which is less well characterized and may have different functions in human and mouse models. Human CD96 interacts with CD155 and ligation of this receptor activates NK cells, while in mice the presence of CD96 correlates with decreased NK cell activation. Mouse CD96 also binds nectin-1, but the effect of this interaction has not yet been determined. Here we show that human nectin-1 directly interacts with CD96 in vitro. The binding site for CD96 is located on the nectin-1 V-domain, which comprises a canonical interface that is shared by nectins to promote cell adhesion. The affinity of nectin-1 for CD96 is lower than for other nectins such as nectin-3 and nectin-1 itself. However, the affinity of nectin-1 for CD96 is similar to its affinity for herpes simplex virus glycoprotein D (HSV gD), which binds the nectin-1 V-domain during virus entry. The affinity of human CD96 for nectin-1 is lower than for its known activating ligand CD155. We also found that human erythroleukemia K562 cells, which are commonly used as susceptible targets to assess NK cell cytotoxicity did not express nectin-1 on their surface and were resistant to HSV infection. When expressed in K562 cells, nectin-1-GFP accumulated at cell contacts and allowed HSV entry. Furthermore, overexpression of nectin-1-GFP led to an increased susceptibility of K562 cells to NK-92 cell cytotoxicity.

Immunization With Fc-Based Recombinant Epstein-Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model.

Epstein-Barr virus (EBV) was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb) and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications.

Role of Microvesicles in the Spread of Herpes Simplex Virus 1 in Oligodendrocytic Cells.

Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in the neurons of sensory ganglia. In some cases, the virus spreads into the central nervous system, causing encephalitis or meningitis. Cells infected with several different types of viruses may secrete microvesicles (MVs) containing viral proteins and RNAs. In some instances, extracellular microvesicles harboring infectious virus have been found. Here we describe the features of shedding microvesicles released by the human oligodendroglial HOG cell line infected with HSV-1 and their participation in the viral cycle. Using transmission electron microscopy, we detected for the first time microvesicles containing HSV-1 virions. Interestingly, the Chinese hamster ovary (CHO) cell line, which is resistant to infection by free HSV-1 virions, was susceptible to HSV-1 infection after being exposed to virus-containing microvesicles. Therefore, our results indicate for the first time that MVs released by infected cells contain virions, are endocytosed by naive cells, and lead to a productive infection. Furthermore, infection of CHO cells was not completely neutralized when virus-containing microvesicles were preincubated with neutralizing anti-HSV-1 antibodies. The lack of complete neutralization and the ability of MVs to infect nectin-1/HVEM-negative CHO-K1 cells suggest a novel way for HSV-1 to spread to and enter target cells. Taken together, our results suggest that HSV-1 could spread through microvesicles to expand its tropism and that microvesicles could shield the virus from neutralizing antibodies as a possible mechanism to escape the host immune response.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons. Extracellular vesicles are a heterogeneous group of membrane vesicles secreted by most cell types. Microvesicles, which are extracellular vesicles which derive from the shedding of the plasma membrane, isolated from the supernatant of HSV-1-infected HOG cells were analyzed to find out whether they were involved in the viral cycle. The importance of our investigation lies in the detection, for the first time, of microvesicles containing HSV-1 virions. In addition, virus-containing microvesicles were endocytosed into CHO-K1 cells and were able to actively infect these otherwise nonpermissive cells. Finally, the infection of CHO cells with these virus-containing microvesicles was not completely neutralized by anti-HSV-1 antibodies, suggesting that these extracellular vesicles might shield the virus from neutralizing antibodies as a possible mechanism of immune evasion.

Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts.

Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor's interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling.

Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells.

Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus--unable to replicate in ICP4-defficient cells--showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells.

Entry mechanisms of herpes simplex virus 1 into murine epidermis: involvement of nectin-1 and herpesvirus entry mediator as cellular receptors.

UNLABELLED: Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin and whether both nectin-1 and HVEM are involved are not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEM has a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence of HVEM can potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo. IMPORTANCE: Herpes simplex virus (HSV) can cause a range of diseases in humans, from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV, and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contributions of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1- or HVEM-deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV-1 entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unraveling HSV invasion of skin, which in turn will allow the development of antiviral reagents.

Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans.

UNLABELLED: Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE: Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally directed against gD, gB, and, to a lesser extent, gC. While several key HSV-neutralizing epitopes within gD and gB are commonly targeted by human serum IgG, others fail to induce consistent responses. These data are particularly relevant to the design of future HSV vaccines.

The effect of cellular differentiation on HSV-1 infection of oligodendrocytic cells.

Herpes simplex type 1 (HSV-1) is a neurotropic virus that infects many types of cells. Previous studies have demonstrated that oligodendrocytic cells are highly susceptible to HSV-1 infection. Here we analysed HSV-1 infection of a human oligodendrocytic cell line, HOG, and oligodendrocyte precursor cells (OPCs) cultured under growth or differentiation conditions. In addition to cell susceptibility, the role of the major cell receptors for viral entry was assessed. Our results revealed that OPCs and HOG cells cultured under differentiation conditions became more susceptible to HSV-1. On the other hand, viral infection induced morphological changes corresponding to differentiated cells, suggesting that HSV-1 might be inducing cell differentiation. We also observed colocalization of HVEM and nectin-1 with viral particles, suggesting that these two major HSV-1 receptors are functional in HOG cells. Finally, electron microscopy assays indicated that HSV-1 may be also entering OLs by macropinocytosis depending on their differentiation stage. In addition, vesicles containing intracellular enveloped virions observed in differentiated cells point to an endocytic mechanism of virus entry. All these data are indicative of diverse entry pathways dependent on the maturation stage of OLs.

Induction of conformational changes at the N-terminus of herpes simplex virus glycoprotein D upon binding to HVEM and nectin-1.

Herpes simplex virus entry is initiated by glycoprotein D (gD) binding to a cellular receptor, such as HVEM or nectin-1. gD is activated by receptor-induced displacement of the C-terminus from the core of the glycoprotein. Binding of HVEM requires the formation of an N-terminal hairpin loop of gD; once formed this loop masks the nectin-1 binding site on the core of gD. We found that HVEM and nectin-1 exhibit non-reciprocal competition for binding to gD. The N-terminus of gD does not spontaneously form a stable hairpin in the absence of receptor and HVEM does not appear to rely on a pre-existing hairpin for binding to gD(3C-38C) mutants. However, HVEM function is affected by mutations that impair optimal hairpin formation. Furthermore, nectin-1 induces a new conformation of the N-terminus of gD. We conclude that the conformation of the N-terminus of gD is actively modified by the direct action of both receptors.