Anti-HSV activity of nectin-1-derived peptides targeting HSV gD: an in-silico and in-vitro approach.

Herpes simplex virus (HSV) infections affect a wide range of the global population. The emergence of resistance to the existing anti-HSV therapy highlights the necessity for an innovative strategy. The interaction of HSV gD with its main host receptor nectin-1 is a potential target for new antiviral drugs. The aim of this study was to develop a peptide derived from nectin-1 targeting HSV gD using the in-silico method and evaluate them for anti-HSV activity. Residues 59-133 of the Nectin-1 V-domain constitute the interaction interface with HSV gD. Bioinformatic tools viz., PEP-FOLD3, ClusPro 2.0, HawkDock and Desmond were used to model the peptide and confirm its binding specificity with HSV gD protein. The peptides with potential interactions were custom synthesized and anti-HSV activity was evaluated in vitro against HSV-1 and HSV-2 by CPE inhibition assay. Five peptide sequences were identified as exhibiting good interaction with HSV-gD proteins. Among them, peptide N1 (residues 76-90) offered maximum protection against HSV-1 (66.57%) and HSV-2 (71.12%) infections. Modification of the identified peptide through peptidomimetic approaches may further enhance the activity and stability of the identified peptide.Communicated by Ramaswamy H. Sarma.

Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function.

Many viruses downregulate their cognate receptors, facilitating virus replication and pathogenesis via processes that are not yet fully understood. In the case of herpes simplex virus 1 (HSV1), the receptor binding protein glycoprotein D (gD) has been implicated in downregulation of its receptor nectin1, but current understanding of the process is limited. Some studies suggest that gD on the incoming virion is sufficient to achieve nectin1 downregulation, but the virus-encoded E3 ubiquitin ligase ICP0 has also been implicated. Here we have used the physiologically relevant nTERT human keratinocyte cell type - which we have previously shown to express readily detectable levels of endogenous nectin1 - to conduct a detailed investigation of nectin1 expression during HSV1 infection. In these cells, nectin1, but not nectin2 or the transferrin receptor, disappeared from the cell surface in a process that required virus protein synthesis rather than incoming virus, but did not involve virus-induced host shutoff. Furthermore, gD was not only required but was sufficient for nectin1 depletion, indicating that no other virus proteins are essential. NK cells were shown to be activated in the presence of keratinocytes, a process that was greatly inhibited in cells infected with wild-type virus. However, degranulation of NK cells was also inhibited in ΔgD-infected cells, indicating that blocking of NK cell activation was independent of gD downregulation of nectin1. By contrast, a superinfection time-course revealed that the ability of HSV1 infection to block subsequent infection of a GFP-expressing HSV1 was dependent on gD and occurred in line with the timing of nectin1 downregulation. Thus, the role of gD-dependent nectin1 impairment during HSV infection is important for virus infection, but not immune evasion, which is achieved by other mechanisms.

Single amino acid mutation of nectin-1 provides remarkable resistance against lethal pseudorabies virus infection in mice.

An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a mouse model with defined point mutation in primary receptor for alphaherpesviruses, nectin-1, by the CRISPR/Cas9 system. It has become clear that phenylalanine at position 129 of nectin-1 is important for binding to viral glycoprotein D (gD), and mutation of phenylalanine 129 to alanine (F129A) prevents nectin-1 binding to gD and virus entry in vitro. Here, to assess the antiviral potential of the single amino acid mutation of nectin-1, F129A, in vivo, we generated genome-edited mutant mouse lines; F129A and 135 knockout (KO). The latter, 135 KO used as a nectin-1 knockout line for comparison, expresses a carboxy-terminal deleted polypeptide consisting of 135 amino acids without phenylalanine 129. In the challenge with 10 LD50 PRV via intranasal route, perfect protection of disease onset was induced by expression of the mutation of nectin-1, F129A (survival rate: 100% in F129A and 135 KO versus 0% in wild type mice). Neither viral DNA/antigens nor pathological changes were detected in F129A, suggesting that viral entry was prevented at the primary site in natural infection. In the challenge with 50 LD50 PRV, lower but still strong protective effect against disease onset was observed (survival rate: 57% in F129A and 75% in 135 KO versus 0% in wild type mice). The present results indicate that single amino acid mutation of nectin-1 F129A provides significant resistance against lethal pseudorabies.

In vitro reconstitution of herpes simplex virus 1 fusion identifies low pH as a fusion co-trigger.

Membrane fusion mediated by herpes simplex virus 1 (HSV-1) is a complex, multi-protein process that is receptor triggered and can occur both at the cell surface and in endosomes. To deconvolute this complexity, we reconstituted HSV-1 fusion with synthetic lipid vesicles in vitro. Using this simplified, controllable system, we discovered that HSV-1 fusion required not only a cognate host receptor but also low pH. On the target membrane side, efficient fusion required cholesterol, negatively charged lipids found in the endosomal membranes, and an optimal balance of lipid order and disorder. On the virion side, the four HSV-1 entry glycoproteins-gB, gD, gH, and gL-were sufficient for fusion. We propose that low pH is a biologically relevant co-trigger for HSV-1 fusion. The dependence of fusion on low pH and endosomal lipids could explain why HSV-1 enters most cell types by endocytosis. We hypothesize that under neutral pH conditions, other, yet undefined, cellular factors may serve as fusion co-triggers. The in vitro fusion system established here can be employed to systematically investigate HSV-1-mediated membrane fusion.IMPORTANCEHSV-1 causes lifelong, incurable infections and diseases ranging from mucocutaneous lesions to fatal encephalitis. Fusion of viral and host membranes is a critical step in HSV-1 infection of target cells that requires multiple factors on both the viral and host sides. Due to this complexity, many fundamental questions remain unanswered, such as the identity of the viral and host factors that are necessary and sufficient for HSV-1-mediated membrane fusion and the nature of the fusion trigger. Here, we developed a simplified in vitro fusion assay to examine the fusion requirements and identified low pH as a co-trigger for virus-mediated fusion in vitro. We hypothesize that low pH has a critical role in cell entry and, potentially, pathogenesis.

AATF/Che-1 RNA polymerase II binding protein overexpression reduces the anti-tumor NK-cell cytotoxicity through activating receptors modulation.

Introduction: AATF/Che-1 over-expression in different tumors is well known and its effect on tumorigenicity is mainly due to its central role demonstrated in the oncogenic pathways of solid tumors, where it controls proliferation and viability. The effect exerted by tumors overexpressing Che-1 on the immune response has not yet been investigated. Methods: Starting from ChIP-sequencing data we confirmed Che-1 enrichment on Nectin-1 promoter. Several co-cultures experiments between NK-cells and tumor cells transduced by lentiviral vectors carrying Che-1-interfering sequence, analyzed by flow-cytometry have allowed a detailed characterization of NK receptors and tumor ligands expression. Results: Here, we show that Che-1 is able to modulate the expression of Nectin-1 ligand at the transcriptional level, leading to the impairment of killing activity of NK-cells. Nectin-1 down-modulation induces a modification in NK-cell ligands expression able to interact with activating receptors and to stimulate NK-cell function. In addition, NK-cells from Che-1 transgenic mice, confirming a reduced expression of activating receptors, exhibit impaired activation and a preferential immature status. Discussion: The critical equilibrium between NK-cell ligand expression on tumor cells and the interaction with NK cell receptors is affected by Che-1 over-expression and partially restored by Che-1 interference. The evidence of a new role for Che-1 as regulator of anti-tumor immunity supports the necessity to develop approaches able to target this molecule which shows a dual tumorigenic function as cancer promoter and immune response modulator.

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.

Corrigendum: High expression of nectin-1 indicates a poor prognosis and promotes metastasis in hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2022.953529.].

High expression of nectin-1 indicates a poor prognosis and promotes metastasis in hepatocellular carcinoma.

Objectives: Nectins are a new class of cell-adhesion molecules that play an important role in tumorigenesis and disease progression. The aim of this study was to investigate the prognostic and pathogenetic roles of nectins in hepatocellular carcinoma (HCC). Methods: The expression levels of the nectin family in HCC and their role in prognosis were analyzed by bioinformatics analysis based on The Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma database. The correlations between nectins and immune cells were analyzed using TIMER. The functional enrichment of the nectin-1 coexpression network was evaluated in TCGA cohort, and the expression levels of nectin-1 were detected by immunohistochemistry and Western blot analysis. A Transwell kit was used for cell migration experiments. Cell proliferation was analyzed using Cell Counting Kit-8. Results: The expression levels of nectin-1 protein in the cancer tissues of 28 patients with HCC were higher than those in paracancerous tissues. The Kaplan-Meier plotter analysis showed that the high expression of all nectin family numbers was related to the poor prognosis of HCC patients. The abnormal expression of nectin-1 effectively distinguished the prognosis at different stages and grades of HCC. The high expression of 17 methylation sites of the nectin-1 gene was related to the high overall survival of HCC patients. Kyoto Encyclopedia of Genes and Genomes analysis of genes negatively correlated with nectin-1, revealing their close relation to the regulation of the immune-effector process. Pearson's correlation analysis showed that nectin-1 was significantly positively correlated with multiple immune genes and B cells, CD4+ T cells, macrophages, neutrophils, and dendritic cell infiltration. Cell proliferation of the knockdown (KD) group decreased significantly compared to the NC-KD group. The number of metastatic cells in the KD group decreased significantly compared to that in the NC-KD group. Conclusions: Abnormal expression of nectins and multiple methylation sites closely correlates with poor prognosis in HCC patients. Nectins are related to immune cell infiltration and immune-related genes. In particular, nectin-1 can promote the proliferation and migration of liver cancer cells and distinguish the prognosis at different stages and grades of HCC. Nectin-1 might be a new potential molecular marker for prognostic evaluation and also a therapeutic target for HCC.

A Nectin1 Mutant Mouse Model Is Resistant to Pseudorabies Virus Infection.

The present study generated nectin1-mutant mice with single amino acid substitution and tested the anti-pseudorabies virus (PRV) ability of the mutant mice, with the aim to establish a model for PRV-resistant livestock. A phenylalanine to alanine transition at position 129 (F129A) of nectin1 was introduced into the mouse genome to generate nectin1 (F129A) mutant mice. The mutant mice were infected with a field-isolated highly virulent PRV strain by subcutaneous injection of virus. We found that the homozygous mutant mice had significantly alleviated disease manifestations and decreased death rate and viral loading in serum and tissue compared with heterozygous mutant and wild-type mice. In addition to disease resistance, the homozygous mutant mice showed a defect in eye development, indicating the side effect on animals by only one amino acid substitution in nectin1. Results demonstrate that gene modification in nectin1 is an effective approach to confer PRV resistance on animals, but the mutagenesis pattern requires further investigation to increase viral resistance without negative effect on animal development.

Pentraxin-3 in the Spinal Dorsal Horn Upregulates Nectin-1 Expression in Neuropathic Pain after Spinal Nerve Damage in Male Mice.

Purpose: Neuropathic pain often originates from nerve injury or diseases of the somatosensory nervous system. However, its specific pathogenesis remains unclear. The requirement for excitatory synaptic plasticity in pain-related syndromes has been demonstrated. A recent study reported that pentraxin-3 is important in glutamatergic synaptic formation and function. Meanwhile, nectin-1 mediates synaptogenesis in neurological disorders. The present study aimed to evaluate whether pentraxin-3 and nectin-1 modulate spinal nerve damage-related neuropathic pain in male mice. Methods: L4 spinal nerve ligation (SNL) in male mice was performed to induce experimental neuropathic pain. Mechanical allodynia and heat hyperalgesia following SNL were based on paw withdrawal (PW) threshold and PW latency, respectively. Spinal pentraxin-3 levels and nectin-1 expression following SNL were examined. Pentraxin-3 and nectin-1 knockdown models were established by the shRNA method. These models were used with a recombinant pentraxin-3 cell model to investigate the underlying mechanisms of SNL. Results: The SNL operation generated persistent decreases in mechanical PW threshold and thermal PW latency, with subsequent long-lasting elevations in spinal pentraxin-3 and nectin-1 expression levels. Pentraxin-3 knockdown reduced SNL-associated neuropathic pain behaviors as well as nectin-1 amounts in the spinal dorsal horn. Nectin-1 deficiency impaired mechanical allodynia and thermal hyperalgesia following spinal nerve injury. The application of recombinant pentraxin-3 in the spinal cord triggered an acute nociception phenotype and induced spinal overexpression of nectin-1. The intrathecal knockdown of nectin-1 prevented exogenous pentraxin-3-evoked pain hypersensitivity. Conclusions: The findings suggest spinal pentraxin-3 is required for SNL-triggered neuropathic pain via nectin-1 upregulation in male mice.

Nectin-1 and Non-muscle Myosin Heavy Chain-IIB: Major Mediators of Herpes Simplex Virus-1 Entry Into Corneal Nerves.

Herpes Simplex Virus 1 (HSV-1) invades corneal nerves upon its infection of the cornea and then establishes latency in the trigeminal ganglion (TG). The latent virus in TG is often reactivated and travels back to the cornea, causing recurrent herpes simplex keratitis (HSK). The entry of HSV-1 into the corneal nerve is considered the initial step of infection resulting in HSV-1 latency and HSK recurrence. Several gD and gB receptors have been identified, including nectin-1, herpes virus entry medium (HVEM) and 3-O-sulfated heparan sulfate (3-OS-HS) as gD receptors, and non-muscle myosin heavy chain IIA (NMHC-IIA), NMHC-IIB and myelin-associated glycoprotein (MAG) as gB receptors. However, which receptors contribute to the entry of HSV-1 into corneal nerves are yet to be determined. This study observed that receptors nectin-1, HVEM, 3-OS-HS, NMHC-IIA, and NMHC-IIB, not MAG, were expressed in healthy corneal nerves. Further, we cultured TG neurons extracted from mice in vitro to screen for functional gD/gB receptors. Both in vitro siRNA knockdown and in vivo antibody blocking of either nectin-1 or NMHC-IIB reduced the entry and the replication of HSV-1 as shown by qPCR analysis and immunofluorescence measure, respectively. Also, we observed that the re-localization and the upregulation expression of NMHC-IIB after HSV-1 exposure were inhibited when gD receptor nectin-1 was knocked down. These data suggest that nectin-1 was the main gD receptor and NMHC-IIB was the main gB receptor in mediating HSV-1 entry and hold promise as therapeutic targets for resolving HSV-1 latency and HSK recurrence.

A potent neutralizing and protective antibody against a conserved continuous epitope on HSV glycoprotein D.

Infections caused by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) remain a serious global health issue, and the medical countermeasures available thus far are limited. Virus-neutralizing monoclonal antibodies (NAbs) are crucial tools for studying host-virus interactions and designing effective vaccines, and the discovery and development of these NAbs could be one approach to treat or prevent HSV infection. Here, we report the isolation of five HSV NAbs from mice immunized with both HSV-1 and HSV-2. Among these were two antibodies that potently cross-neutralized both HSV-1 and HSV-2 with the 50% virus-inhibitory concentrations (IC50) below 200 ng/ml, one of which (4A3) exhibited high potency against HSV-2, with an IC50 of 59.88 ng/ml. 4A3 neutralized HSV at the prebinding stage and prevented HSV infection and cell-to-cell spread. Significantly, administration of 4A3 completely prevented weight loss and improved survival of mice challenged with a lethal dose of HSV-2. Using structure-guided molecular modeling combined with alanine-scanning mutagenesis, we observed that 4A3 bound to a highly conserved continuous epitope (residues 216 to 220) within the receptor-binding domain of glycoprotein D (gD) that is essential for viral infection and the triggering of membrane fusion. Our results provide guidance for developing NAb drugs and vaccines against HSV.

Susceptibility of Human and Murine Dermal Fibroblasts to Herpes Simplex Virus 1 in the Absence and Presence of Extracellular Matrix.

Herpes simplex virus 1 (HSV-1) invades its human host via the skin and mucosa and initiates infection in the epithelium. While human and murine epidermis are highly susceptible to HSV-1, we recently observed rare infected cells in the human dermis and only minor infection efficiency in murine dermis upon ex vivo infection. Here, we investigated why cells in the dermis are so inefficiently infected and explored potential differences between murine and human dermal fibroblasts. In principle, primary fibroblasts are highly susceptible to HSV-1; however, we found a delayed infection onset in human compared to murine cells. Intriguingly, only a minor delayed onset of infection was evident in collagen-embedded compared to unembedded human fibroblasts, although expression of the receptor nectin-1 dropped after collagen embedding. This finding is in contrast to previous observations with murine fibroblasts where collagen embedding delayed infection. The application of latex beads revealed limited penetration in the dermis, which was more pronounced in the human than in the murine dermis, supporting the species-specific differences already observed for HSV-1 invasion. Our results suggest that the distinct organization of human and murine dermis contributes to the presence and accessibility of the HSV-1 receptors as well as to the variable barrier function of the extracellular matrix. These contributions, in turn, give rise to inefficient viral access to cells in the dermis while dermal fibroblasts in culture are well infected. IMPORTANCE Dermal fibroblasts are exposed to HSV-1 upon invasion in skin during in vivo infection. Thus, fibroblasts represent a widely used experimental tool to understand virus-host cell interactions and are highly susceptible in culture. The spectrum of fibroblasts' characteristics in their in vivo environment, however, clearly differs from the observations under cell culture conditions, implying putative variations in virus-cell interactions. This becomes evident when ex vivo infection studies in murine as well as human dermis revealed the rather inefficient penetration of HSV-1 in the tissue and uptake in the dermal fibroblasts. Here, we initiated studies to explore the contributions of receptor presence and accessibility to efficient infection of dermal fibroblasts. Our results strengthen the heterogeneity of murine and human dermis and imply that the interplay between dermal barrier function and receptor presence determine how well HSV-1 penetrates the dermis.

Nectin-1 Is an Entry Mediator for Varicella-Zoster Virus Infection of Human Neurons.

Varicella-zoster virus (VZV) maintains lifelong latency in neurons following initial infection and can subsequently be reactivated to result in herpes zoster or severe neurological manifestations such as encephalitis. Mechanisms of VZV neuropathogenesis have been challenging to study due to the strict human tropism of the virus. Although neuronal entry mediators of other herpesviruses, including herpes simplex virus, have been identified, little is known regarding how VZV enters neurons. Here, we utilize a human stem cell-based neuronal model to characterize cellular factors that mediate entry. Through transcriptional profiling of infected cells, we identify the cell adhesion molecule nectin-1 as a candidate mediator of VZV entry. Nectin-1 is highly expressed in the cell bodies and axons of neurons. Either knockdown of endogenous nectin-1 or incubation with soluble forms of nectin-1 produced in mammalian cells results in a marked decrease in infectivity of neurons. Notably, while addition of soluble nectin-1 during viral infection inhibits infectivity, addition after infection has no effect on infectivity. Ectopic expression of human nectin-1 in a cell line resistant to productive VZV infection confers susceptibility to infection. In summary, we have identified nectin-1 as a neuronal entry mediator of VZV. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox, gains access to neurons during primary infection where it resides lifelong, and can later be reactivated. Reactivation is associated with shingles and postherpetic neuralgia, as well as with severe neurologic complications, including vasculitis and encephalitis. Although the varicella vaccine substantially decreases morbidity and mortality associated with primary infection, the vaccine cannot prevent the development of neuronal latency, and vaccinated populations are still at risk for reactivation. Furthermore, immunocompromised individuals are at higher risk for VZV reactivation and associated complications. Little is known regarding how VZV enters neurons. Here, we identify nectin-1 as an entry mediator of VZV in human neurons. Identification of nectin-1 as a neuronal VZV entry mediator could lead to improved treatments and preventative measures to reduce VZV related morbidity and mortality.

A monoclonal antibody neutralizes pesudorabies virus by blocking gD binding to the receptor nectin-1.

Pseudorabies virus (PRV) was isolated from some human cases recently and the infected patients manifested respiratory dysfunction and acute neurological symptoms. However, no effective drug or vaccine, preventing the progression of PRV infection, is available. Nectin-1 was the only reported receptor for PRV cell entry both swine and human origin, representing an excellent target to block PRV infection, and especially its transmission from pigs to humans. A PRV-gD specific mAbs (10B6) was isolated from hybridomas and its neutralizing activities in vitro and in vivo were determined. 10B6 exhibited effective neutralizing activities in vitro with IC50 = 2.514 µg/ml and 4.297 µg/ml in the presence and absence of complement. And in vivo, 10B6 provided 100% protection against PRV lethal challenge with a dose of 15 mg/kg. Further, 10B6 could bind to a conserved epitope, 316QPAEPFP322, locating in gD pro-fusion domain, and finally blocks the binding of PRV-gD to nectin-1. Moreover, 10B6 showed an effective inhibition on PRV cell-attachment in a cell type-independent manner and could also block the virus spreading among cells. 10B6 exhibited effectively neutralizing activities to Chinese PRV variant strain in vitro and in vivo by blocking gD binding to nectin-1, implied both prophylactic and therapeutic interventions against PRV infections.

Ex Vivo Infection of Human Skin with Herpes Simplex Virus 1 Reveals Mechanical Wounds as Insufficient Entry Portals via the Skin Surface.

Herpes simplex virus 1 (HSV-1) enters its human host via the skin and mucosa. The open question is how the virus invades this highly protective tissue in vivo to approach its receptors in the epidermis and initiate infection. Here, we performed ex vivo infection studies in human skin to investigate how susceptible the epidermis and dermis are to HSV-1 and whether wounding facilitates viral invasion. Upon ex vivo infection of complete skin, only sample edges with integrity loss demonstrated infected cells. After removal of the dermis, HSV-1 efficiently invaded the basal layer of the epidermis and, from there, gained access to suprabasal layers. This finding supports a high susceptibility of all epidermal layers which correlated with the surface expression of the receptors nectin-1 and herpesvirus entry mediator (HVEM). In contrast, only single infected cells were detected in the separated dermis, where minor expression of the receptors was found. Interestingly, after wounding, nearly no infection of the epidermis was observed via the skin surface. However, if the wounding of the skin samples led to breaks through the dermis, HSV-1 infected mainly keratinocytes via the damaged dermal layer. The application of latex beads revealed only occasional entry via the wounded dermis; however, it facilitated penetration via the wounded skin surface. Thus, we suggest that although the wounded human skin surface allows particle penetration, the skin still provides barriers that prevent HSV-1 from reaching its receptors. IMPORTANCE The human pathogen herpes simplex virus 1 (HSV-1) invades its host via the skin and mucosa, which leads to primary infection of the epithelium. As the various epithelial barriers effectively protect the tissue against viral invasion, successful infection most likely depends on tissue damage. We addressed the initial invasion process in human skin by ex vivo infection to understand how HSV-1 overcomes physical skin barriers and reaches its receptors to enter skin cells. Our results demonstrate that intact skin samples allow viral access only from the edges, while the epidermis is highly susceptible once the basal epidermal layer serves as an initial entry portal. Surprisingly, mechanical wounding did not facilitate HSV-1 entry via the skin surface, although latex beads still penetrated via the lesions. Our results imply that successful invasion of HSV-1 depends on how well the virus can reach its receptors, which was not accomplished by skin lesions under ex vivo conditions.

Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo.

Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus, is approved for intralesional injection of unresectable stage IIIB/IVM1a melanoma. However, it is still unclear which parameter(s) predict treatment response or failure. Our study aimed at characterizing surface receptors Nectin-1 and the herpes virus entry mediator (HVEM) in addition to intracellular molecules cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) as potential bio-markers for oncolytic virus treatment. In 20 melanoma cell lines, oncolytic activity of T-VEC was correlated with the expression of Nectin-1 but not HVEM, as evaluated via flow cytometry and immunohistochemistry. Knockout using CRISPR/Cas9 technology confirmed the superior role of Nectin-1 over HVEM for entry and oncolytic activity of T-VEC. Neither cGAS nor STING as evaluated by Western Blot and immunohistochemistry correlated with T-VEC induced oncolysis. The role of these biomarkers was retrospectively analyzed for the response of 35 cutaneous melanoma metastases of 21 patients to intralesional T-VEC injection, with 21 (60.0%) of these lesions responding with complete (n = 16) or partial regression (n = 5). Nectin-1 expression in pretreatment biopsies significantly predicted treatment outcome, while the expression of HVEM, cGAS, and STING was not prognostic. Altogether, Nectin-1 served as biomarker for T-VEC-induced melanoma regression in vitro and in vivo.

Oncolytic herpes simplex virus infects myeloma cells in vitro and in vivo.

Because most patients with multiple myeloma (MM) develop resistance to current regimens, novel approaches are needed. Genetically modified, replication-competent oncolytic viruses exhibit high tropism for tumor cells regardless of cancer stage and prior treatment. Receptors of oncolytic herpes simplex virus 1 (oHSV-1), NECTIN-1, and HVEM are expressed on MM cells, prompting us to investigate the use of oHSV-1 against MM. Using oHSV-1-expressing GFP, we found a dose-dependent increase in the GFP+ signal in MM cell lines and primary MM cells. Whereas NECTIN-1 expression is variable among MM cells, we discovered that HVEM is ubiquitously and highly expressed on all samples tested. Expression of HVEM was consistently higher on CD138+/CD38+ plasma cells than in non-plasma cells. HVEM blocking demonstrated the requirement of this receptor for infection. However, we observed that, although oHSV-1 could efficiently infect and kill all MM cell lines tested, no viral replication occurred. Instead, we identified that oHSV-1 induced MM cell apoptosis via caspase-3 cleavage. We further noted that oHSV-1 yielded a significant decrease in tumor volume in two mouse xenograft models. Therefore, oHSV-1 warrants exploration as a novel potentially effective treatment option in MM, and HVEM should be investigated as a possible therapeutic target.

NOTCH Signaling Controls Ciliary Body Morphogenesis and Secretion by Directly Regulating Nectin Protein Expression.

Anterior segment dysgenesis is often associated with cornea diseases, cataracts, and glaucoma. In the anterior segment, the ciliary body (CB) containing inner and outer ciliary epithelia (ICE and OCE) secretes aqueous humor that maintains intraocular pressure (IOP). However, CB development and function remain poorly understood. Here, this study shows that NOTCH signaling in the CB maintains the vitreous, IOP, and eye structures by regulating CB morphogenesis, aqueous humor secretion, and vitreous protein expression. Notch2 and Notch3 function via RBPJ in the CB to control ICE-OCE adhesion, CB morphogenesis, aqueous humor secretion, and protein expression, thus maintaining IOP and eye structures. Mechanistically, NOTCH signaling transcriptionally controls Nectin1 expression in the OCE to promote cell adhesion for driving CB morphogenesis and to directly stabilize Cx43 for controlling aqueous humor secretion. Finally, NOTCH signaling directly controls vitreous protein secretion in the ICE. Therefore, this study provides important insight into CB functions and involvement in eye diseases.

EpCAM is essential for maintenance of the small intestinal epithelium architecture via regulation of the expression and localization of proteins that compose adherens junctions.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in mammalian intestines, and is essential for maintaining the homeostasis of the intestinal epithelium. EpCAM protein is localized at tight junctions and the basolateral membrane of the intestinal epithelium, where it interacts with many cell adhesion molecules. To explore the molecular functions of EpCAM in regulating adherens junctions in the intestinal epithelium, EpCAM knockout embryos and newborn pups were analyzed. Hematoxylin and eosin staining was used to assess the histology of the duodenum, jejunum, ileum and colon from wild-type and EpCAM­/­ mice at E18.5, P0 and P3. The expression and localization of adherens junction­associated genes and genes that encode the proteins that participate in the assembly of adherens junctions were measured at the mRNA and protein levels using qPCR, western blot analysis and immunofluorescence staining. The results showed that although there was no significant damage to the intestines of EpCAM­/­ mice at E18.5 and P0, they were significantly damaged at P3 in mutant mice. The expression of adherens junction­associated genes in EpCAM mutant mice was normal at the mRNA level from E18.5 to P3, but their protein levels were gradually reduced and mislocalized from E18.5 to P3. The expression of nectin 1, which can regulate the assembly and adhesion activity of E­cadherin, was also gradually reduced at both the mRNA and protein levels in the intestinal epithelium of EpCAM mutant mice from E18.5 to P3. In summary, the loss of EpCAM may cause the reduction and mislocalization of proteins that compose adherens junctions partly via the downregulation of nectin 1 in the intestines.