Assessing the binding of nectin-1 and CD96 in human cells to investigate how herpes simplex virus evades the innate immune system

Nectin-1 is an adhesion protein that also serves as a receptor for herpes simplex virus (HSV) to enter cells. In epithelial tissues nectin-1 proteins naturally bind to other nectin-1 molecules on adjacent cells to organize intercellular junctions. Recently, our lab showed that human nectin-1 also binds to the natural killer (NK) cell receptor CD96 (Holmes et al, PloS One, 2019). CD96 modulates the immune response of NK cells, which are part of the innate immune system of mammals. NK cells play an important role in eliminating virally infected cells and cancer cells. When HSV infection occurs, nectin-1 located at the cell surface becomes internalized by endocytosis. We hypothesize that HSV down-regulates nectin-1 from the surface of infected cells to prevent their detection and destruction by NK cells. To study how nectin-1 and CD96 proteins bind to each other at cell contacts, cell lines expressing fluorescently tagged nectin-1 and CD96 are being generated. Previously, we used mouse melanoma B78H1 cells as a model which are normally deficient in both nectin-1 and CD96. B78H1 cells were transfected to stably express fluorescent human nectin-1 (hNectin-1GFP, green), however expression of fluorescent human CD96 (hCD96mCherry, red) appears to be unstable in these cells. Here, human cell lines 293 and K562 are being used. 293 cells are robust human embryonic kidney cells that are being used as a positive control for transfection. K562 cells are human myelogenous leukemia cells that have been shown previously in this lab to be deficient in nectin-1 and CD96. We are currently attempting selection of clonal cell lines for these constructs by fluorescent microscopy and flow cytometry. The cells that express fluorescent CD96 and nectin-1 will be mixed in co-cultures to observe co-localization of nectin-1 and CD96 at cell contacts. This approach will provide a unique setting to investigate and understand how HSV affects the NK cell innate immune response

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 (NPC) 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 to 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 and binding to its receptor by ELISA and SPR. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared to gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent neutralizing antibodies 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. Please click Additional Files below to see the full abstract

Lessons From the Pandemic: Engaging Wicked Problems With Transdisciplinary Deliberation

Some crises, such as those brought on or exposed by the COVID-19 pandemic, are wicked problems—large, complex problems with no immediate answer. As such, they make rich centerpieces for learning with respect to public deliberation and issue-based dialogue. This essay reflects on an experimental, transdisciplinary health and science communication course entitled Comprehending COVID-19. The course represents a collaborative effort among 14 faculty representing 10 different academic departments to create a resource for teaching students how to deliberate the pandemic, despite its attending, oversaturated, fake-news-infused, infodemic. We offer transdisciplinary deliberation as a pedagogical framework to expand communication repertoires in ways useful for sifting through the messiness of an infodemic while also developing key deliberation skills for productively engaging participatory decision-making with concern to wicked problems

Cytolethal Distending Toxin-Induced Cell Cycle Arrest of Lymphocytes is Dependent Upon Recognition and Binding to Cholesterol

Induction of cell cycle arrest in lymphocytes after exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. In this study we further demonstrate that the association of Cdt with lymphocyte plasma membranes is dependent upon binding to cholesterol. Depletion of cholesterol resulted in reduced toxin binding, whereas repletion of cholesterol-depleted cells restored binding. We employed fluorescence resonance energy transfer and surface plasmon resonance to demonstrate that toxin association with model membranes is dependent upon the concentration of cholesterol; moreover, these interactions were cholesterol-specific as the toxin failed to interact with model membranes containing stigmasterol, ergosterol, or lanosterol. Further analysis of the toxin indicated that the CdtC subunit contains a cholesterol recognition/interaction amino acid consensus (CRAC) region. Mutation of the CRAC site resulted in decreased binding of the holotoxin to cholesterol-containing model membranes as well as to the surface of Jurkat cells. The mutant toxin also exhibited reduced capacity for intracellular transfer of the active toxin subunit, CdtB, as well as reduced toxicity. Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for Cdt and that this association can be blocked by either depleting membranes of cholesterol or mutation of the CRAC site. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc

α-Herpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites

α-Herpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. We report that in vitro infection of swine TG neurons with the homologous swine α-herpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Nonneuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope glycoprotein D (gD) was found to be necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 mitogen-activated protein kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of α-herpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with signaling events and neuronal changes that may prepare efficient egress of progeny virus

A key role for nectin-1 in the ventral hippocampus in contextual fear memory

Nectins are cell adhesion molecules that are widely expressed in the brain. Nectin expression shows a dynamic spatiotemporal regulation, playing a role in neural migratory processes during development. Nectin-1 and nectin-3 and their heterophilic trans-interactions are important for the proper formation of synapses. In the hippocampus, nectin-1 and nectin-3 localize at puncta adherentia junctions and may play a role in synaptic plasticity, a mechanism essential for memory and learning. We evaluated the potential involvement of nectin-1 and nectin-3 in memory consolidation using an emotional learning paradigm. Rats trained for contextual fear conditioning showed transient nectin-1—but not nectin-3—protein upregulation in synapse-enriched hippocampal fractions at about 2 h posttraining. The upregulation of nectin-1 was found exclusively in the ventral hippocampus and was apparent in the synaptoneurosomal fraction. This upregulation was induced by contextual fear conditioning but not by exposure to context or shock alone. When an antibody against nectin-1, R165, was infused in the ventral-hippocampus immediately after training, contextual fear memory was impaired. However, treatment with the antibody in the dorsal hippocampus had no effect in contextual fear memory formation. Similarly, treatment with the antibody in the ventral hippocampus did not interfere with acoustic memory formation. Further control experiments indicated that the effects of ventral hippocampal infusion of the nectin-1 antibody in contextual fear memory cannot be ascribed to memory non-specific effects such as changes in anxiety-like behavior or locomotor behavior. Therefore, we conclude that nectin-1 recruitment to the perisynaptic environment in the ventral hippocampus plays an important role in the formation of contextual fear memories. Our results suggest that these mechanisms could be involved in the connection of emotional and contextual information processed in the amygdala and dorsal hippocampus, respectively, thus opening new venues for the development of treatments to psychopathological alterations linked to impaired contextualization of emotions

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 OLsThe work was partially supported by the Ministerio de Economía y Competitividad-MINECO (SAF2012-40023 and RD12-0032-12 -partially funded by FEDER- European Union/Una manera de hacer Europa). F de C is hired by Gobierno de Castilla-La Mancha-SESCAM. CK is supported by Public Health Service grant AI-097171 from the National Institute of Allergy and Infectious Disease

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