Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion.

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human embryonic kidney (HEK293T) cells has been shown to be a cholesterol-rich, lipid raft-dependent process. In this study, we investigated if the presence of a cholesterol uptake receptor Niemann-pick type c1-like1 (NPC1L1) impacts SARS-CoV-2 cell entry. Initially, we utilized reporter-based pseudovirus cell entry assays and a spike (S) glycoprotein-mediated cell-to-cell fusion assay. Using Chinese hamster ovary (CHO-K1) cells, which lack endogenous receptors for SARS-CoV-2 entry, our data showed that the co-expression of NPC1L1 together with the ACE2 receptor synergistically increased SARS-CoV-2 pseudovirus entry even more than the cells expressing ACE-2 receptor alone. Similar results were also found with the HEK293T cells endogenously expressing the ACE2 receptor. Co-cultures of effector cells expressing S glycoprotein together with target cells co-expressing ACE-2 receptor with NPC1L1 significantly promoted quantitative cell-to-cell fusion, including syncytia formation. Finally, we substantiated that an elevated expression of NPC1L1 enhanced entry, whereas the depletion of NPC1L1 resulted in a diminished SARS-CoV-2 entry in HEK293T-ACE2 cells using authentic SARS-CoV-2 virus in contrast to their respective control cells. Collectively, these findings underscore the pivotal role of NPC1L1 in facilitating the cellular entry of SARS-CoV-2. Importance: Niemann-Pick type C1-like1 (NPC1L1) is an endosomal membrane protein that regulates intracellular cholesterol trafficking. This protein has been demonstrated to play a crucial role in the life cycle of several clinically important viruses. Although SARS-CoV-2 exploits cholesterol-rich lipid rafts as part of its viral entry process, the role of NPC1L1 in SARS-CoV-2 entry remains unclear. Our research represents the first-ever demonstration of NPC1L1's involvement in facilitating SARS-CoV-2 entry. The observed role of NPC1L1 in human kidney cells is not only highly intriguing but also quite relevant. This relevance stems from the fact that NPC1L1 exhibits high expression levels in several organs, including the kidneys, and the fact that kidney damages are reported during severe cases of SARS-CoV-2. These findings may help us understand the new functions and mechanisms of NPC1L1 and could contribute to the identification of new antiviral targets.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) Enhances ATP Production in B Cell Tumors through mTOR and HIF-1α.

Epstein-Barr Virus (EBV) exists in a latent state in 90% of the world's population and is linked to numerous cancers, such as Burkitt's Lymphoma, Hodgkin's, and non-Hodgkin's Lymphoma. One EBV latency protein, latency membrane protein 2A (LMP2A), is expressed in multiple latency phenotypes. LMP2A signaling has been extensively studied and one target of LMP2A is the mammalian target of rapamycin (mTOR). Since mTOR has been linked to reprogramming tumor metabolism and increasing levels of hypoxia-inducible factor 1 α (HIF-1α), we hypothesized that LMP2A would increase HIF-1α levels to enhance ATP generation in B lymphoma cell lines. Our data indicate that LMP2A increases ATP generation in multiple Burkitt lymphoma cell lines that were dependent on HIF-1α. Subsequent studies indicate that the addition of the mTOR inhibitor, rapamycin, blocked the LMP2A-dependent increase in HIF-1α. Further studies demonstrate that LMP2A does not increase HIF-1α levels by increasing HIF-1α RNA or STAT3 activation. In contrast, LMP2A and mTOR-dependent increase in HIF-1α required mTOR-dependent phosphorylation of p70 S6 Kinase and 4E-BP1. These findings implicate the importance of LMP2A in promoting B cell lymphoma survival by increasing ATP generation and identifying potential pharmaceutical targets to treat EBV-associated tumors.

The impact of Epstein-Barr virus latent membrane protein 2A on the production of B cell activating factor of the tumor necrosis factor family (BAFF), APRIL and their receptors.

INTRODUCTION: Epstein-Barr virus (EBV) establishes a lifelong infection in human B cells where the virus consistently expresses Latent Membrane Protein 2A (LMP2A) to promote B cell survival. A prior study indicates that LMP2A may increase the production of the pro-survival factor, B cell Activating Factor of the tumor necrosis factor family (BAFF), which could also indirectly increase B cell survival. The current study sought to extend these findings and determine if LMP2A increased BAFF production and/or the responsiveness of LMP2A-expressing cells to this cytokine. METHODS: Four independently derived LMP2A-negative and -positive B cell lymphoma cell lines were analyzed for BAFF and APRIL levels by both ELISA and Western Blot analysis. Additionally, flow cytometric analysis measured any LMP2A-dependent changes in the receptors for BAFF and APRIL (BAFF-R, transmembrane activator and calcium-modulator and cyclophilin ligand interactor [TACI], B cell maturation antigen [BCMA]) in both LMP2A-negative and -positive B cell lymphoma cell lines. RESULTS: In contrast to previous reports, our data indicate that LMP2A does not increase the expression of BAFF or APRIL by Western blot analysis or ELISA. Additionally, flow cytometric analysis indicates that LMP2A does not influence the expression of the receptors for BAFF and APRIL: TACI, BAFF-R, and BCMA. CONCLUSION: Therefore, these data suggest that while EBV utilizes other latency proteins to regulate BAFF production, EBV does not appear to use LMP2A to enhance BAFF-or APRIL-dependent survival to promote EBV latency.

Lipopolysaccharide from the Cyanobacterium Geitlerinema sp. Induces Neutrophil Infiltration and Lung Inflammation.

Glucocorticoid-resistant asthma, which predominates with neutrophils instead of eosinophils, is an increasing health concern. One potential source for the induction of neutrophil-predominant asthma is aerosolized lipopolysaccharide (LPS). Cyanobacteria have recently caused significant tidal blooms, and aerosolized cyanobacterial LPS has been detected near the cyanobacterial overgrowth. We hypothesized that cyanobacterial LPS contributes to lung inflammation by increasing factors that promote lung inflammation and neutrophil recruitment. To test this hypothesis, c57Bl/6 mice were exposed intranasally to LPS from the cyanobacterium member, Geitlerinema sp., in vivo to assess neutrophil infiltration and the production of pro-inflammatory cytokines and chemokines from the bronchoalveolar fluid by ELISA. Additionally, we exposed the airway epithelial cell line, A549, to Geitlerinema sp. LPS in vitro to confirm that airway epithelial cells were stimulated by this LPS to increase cytokine production and the expression of the adhesion molecule, ICAM-1. Our data demonstrate that Geitlerinema sp. LPS induces lung neutrophil infiltration, the production of pro-inflammatory cytokines such as Interleukin (IL)-6, Tumor necrosis factor-alpha, and Interferongamma as well as the chemokines IL-8 and RANTES. Additionally, we demonstrate that Geitlerinema sp. LPS directly activates airway epithelial cells to produce pro-inflammatory cytokines and the adhesion molecule, Intercellular Adhesion Molecule-1 (ICAM-1), in vitro using the airway epithelial cell line, A549. Based on our findings that use Geitlerinema sp. LPS as a model system, the data indicate that cyanobacteria LPS may contribute to the development of glucocorticoid-resistant asthma seen near water sources that contain high levels of cyanobacteria.

American Association of Immunologists Recommendations for an Undergraduate Course in Immunology.

Identifying the "essential" components of an undergraduate immunology lecture course can be daunting because of the varying postgraduate pathways students take. The American Association of Immunologists Education Committee commissioned an Ad Hoc Committee, representing undergraduate, graduate, and medical institutions as well as the biotechnology community, to develop core curricular recommendations for teaching immunology to undergraduates. In a reiterative process involving the American Association of Immunologists teaching community, 14 key topics were identified and expanded to include foundational concepts, subtopics and examples, and advanced subtopics, providing a flexible list for curriculum development and avenues for higher-level learning. Recommendations for inclusive and antiracist teaching that outline opportunities to meet the needs of diverse student populations were also developed. The consensus recommendations can be used to accommodate various course settings and will bridge undergraduate and graduate teaching and prepare diverse students for subsequent careers in the biomedical field.

Preferential recognition and antagonism of SARS-CoV-2 spike glycoprotein binding to 3-O-sulfated heparan sulfate.

The COVID-19 pandemic caused by SARS-CoV-2 is in immediate need of an effective antidote. Although the Spike glycoprotein (SgP) of SARS-CoV-2 has been shown to bind to heparins, the structural features of this interaction, the role of a plausible heparan sulfate proteoglycan (HSPG) receptor, and the antagonism of this pathway through small molecules remain unaddressed. Using an in vitro cellular assay, we demonstrate HSPGs modified by the 3-O-sulfotransferase isoform-3, but not isoform-5, preferentially increased SgP-mediated cell-to-cell fusion in comparison to control, unmodified, wild-type HSPGs. Computational studies support preferential recognition of the receptor-binding domain of SgP by 3-O-sulfated HS sequences. Competition with either fondaparinux, a 3-O-sulfated HS-binding oligopeptide, or a synthetic, non-sugar small molecule, blocked SgP-mediated cell-to-cell fusion. Finally, the synthetic, sulfated molecule inhibited fusion of GFP-tagged pseudo SARS-CoV-2 with human 293T cells with sub-micromolar potency. Overall, overexpression of 3-O-sulfated HSPGs contribute to fusion of SARS-CoV-2, which could be effectively antagonized by a synthetic, small molecule.

Discovering small-molecule therapeutics against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global health pandemic. The lack of effective treatments, coupled with its etiology, has resulted in more than 400,000 deaths at the time of writing. The SARS-CoV-2 genome is highly homologous to that of SARS-CoV, the causative agent behind the 2003 SARS outbreak. Based on prior reports, clinicians have pursued the off-label use of several antiviral drugs, while the scientific community has responded by seeking agents against traditional targets, especially viral proteases. However, several avenues remain unexplored, including disrupting E and M protein oligomerization, outcompeting host glycan-virus interactions, interfering with the heparan sulfate proteoglycans-virus interaction, and others. In this review, we highlight some of these opportunities while summarizing the drugs currently in use against coronavirus 2019 (COVID-19).

Biochemical and Functional Analysis of Cyanobacterium Geitlerinema sp. LPS on Human Monocytes.

Cyanobacterial blooms are an increasing source of environmental toxins that affect both human and animals. After ingestion of cyanobacteria, such as Geitlerinema sp., toxins and lipopolysaccharide (LPS) from this organism induce fever, gastrointestinal illness, and even death. However, little is known regarding the effects of cyanobacterial LPS on human monocytes after exposure to LPS upon ingestion. Based on our previous data using Geitlerinema sp. LPS (which was previously named Oscillatoria sp., a genus belonging to the same order as Geitlerinema), we hypothesized that Geitlerinema sp. LPS would activate human monocytes to proliferate, phagocytose particles, and produce cytokines that are critical for promoting proinflammatory responses in the gut. Our data demonstrate that Geitlerinema sp. LPS induced monocyte proliferation and TNF-α, IL-1, and IL-6 production at high concentrations. In contrast, Geitlerinema sp. LPS is equally capable of inducing monocyte-mediated phagocytosis of FITC-latex beads when compared with Escherichia coli LPS, which was used as a positive control for our experiments. In order to understand the mechanism responsible for the difference in efficacy between Geitlerinema sp. LPS and E. coli LPS, we performed biochemical analysis and identified that Geitlerinema sp. LPS was composed of significantly different sugars and fatty acid side chains in comparison to E. coli LPS. The lipid A portion of Geitlerinema sp. LPS contained longer fatty acid side chains, such as C15:0, C16:0, and C18:0, instead of C12:0 found in E. coli LPS which may explain the decreased efficacy and toxicity of Geitlerinema sp. LPS in comparison to E. coli LPS.

Epstein-Barr virus LMP2A utilizes Syk and PI3K to activate NF-κB in B-cell lymphomas to increase MIP-1α production.

The incidence of Hodgkin's lymphoma (HL) is growing due to an increase in Epstein-Barr virus (EBV)-associated HL in AIDS patients. The HL tumor microenvironment is vital for the survival of the malignant Hodgkin-Reed Sternberg (HRS) cells of HL, which express the EBV protein latent membrane protein 2A (LMP2A). While previous work shows that LMP2A mimics B-cell receptor (BCR) signaling to promote the survival of HRS cells, the ability of LMP2A to establish and maintain the tumor microenvironment through the production of chemokines remains unknown. Since BCR signaling induces the production of the chemokine macrophage inflammatory protein-1α (MIP-1α), and since LMP2A is a BCR mimic, we hypothesized that LMP2A increases MIP-1α levels. A comparison of multiple LMP2A-negative and -positive cell lines demonstrates that LMP2A increases MIP-1α. Additionally, LMP2A-mutant cell lines and pharmacologic inhibitors indicate that LMP2A activates a Syk/PI3K/NF-κB pathway to enhance MIP-1α. Finally, based on the finding that an NF-κB inhibitor decreased MIP-1α RNA/protein in LMP2A-positive cells, we are the first to demonstrate that LMP2A increases the nuclear localization of the NF-κB p65 subunit using DNA-binding assays and confocal microscopy in human B cells. These findings not only have implications for the treatment of HL, but also other LMP2A-expressing B-cell tumors that overexpress NF-κB.

Effects of cyanobacteria Oscillatoria sp. lipopolysaccharide on B cell activation and Toll-like receptor 4 signaling.

Cyanobacteria ("blue-green algae"), such as Oscillatoria sp., are a ubiquitous group of bacteria found in freshwater systems worldwide that are linked to illness and in some cases, death among humans and animals. Exposure to cyanobacteria occurs via ingestion of contaminated water or food-products. Exposure of the gut to these bacteria also exposes their toxins, such as lipopolysaccharide (LPS), to B cells in the gut associated lymphoid tissue. However, the effect of Oscillatoria sp. LPS on B cell activation is unknown. To test the hypothesis that Oscillatoria sp. LPS exposure to murine B cells would result in B cell activation, murine B cells were incubated in the absence or presence of Oscillatoria sp. LPS or E. coli LPS as a positive control. The data indicate that Oscillatoria sp. LPS induces B cells to proliferate, upregulate MHC II and CD86, enhance antigen uptake and induce IgM production at low levels. Additional studies demonstrate that this low level of stimulation may be due to incomplete TLR4 signaling induced by Oscillatoria sp. LPS, since IRF-3 is not induced in B cells after stimulation with Oscillatoria sp. LPS. These findings have important implications for the mechanisms of toxicity of cyanobacteria in both humans and animals.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 production through the activation of Bruton's tyrosine kinase and STAT3.

Previous data demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 to promote the survival of LMP2A-expressing B cell lymphomas. Since STAT3 is an important regulator of IL-10 production, we hypothesized that LMP2A activates a signal transduction cascade that increases STAT3 phosphorylation to enhance IL-10. Using LMP2A-negative and -positive B cell lines, the data indicate that LMP2A requires the early signaling molecules of the Syk/RAS/PI3K pathway to increase IL-10. Additional studies indicate that the PI3K-regulated kinase, BTK, is responsible for phosphorylating STAT3, which ultimately mediates the LMP2A-dependent increase in IL-10. These data are the first to show that LMP2A signaling results in STAT3 phosphorylation in B cells through a PI3K/BTK-dependent pathway. With the use of BTK and STAT3 inhibitors to treat B cell lymphomas in clinical trials, these findings highlight the possibility of using new pharmaceutical approaches to treat EBV-associated lymphomas that express LMP2A.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A)-mediated changes in Fas expression and Fas-dependent apoptosis: Role of Lyn/Syk activation.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

Epstein-Barr virus LMP2A increases IL-10 production in mitogen-stimulated primary B-cells and B-cell lymphomas.

Epstein-Barr virus (EBV) latently infected B-cells are the precursors of EBV-associated malignancies. EBV-infection induces the production of pro-survival and anti-inflammatory cytokines that may be important in the transition between latency and malignancy. One EBV protein, LMP2A, can be detected in both latently infected resting B-cells and in EBV-associated malignancies. Therefore, we tested the ability of LMP2A to influence cytokine production using both LMP2A-Tg primary B-cells and LMP2A-expressing B-cell lines. Our data demonstrate that LMP2A does not globally alter B-cell-produced cytokine levels, but specifically targets IL-10. Additional studies using ELISA and real-time-RT-PCR confirm that LMP2A utilizes PI3-kinase to increase IL-10 levels. Finally, the data demonstrate that LMP2A-expressing B-cell lines are more dependent on IL-10 for survival in comparison to LMP2A-negative B-cell lines. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumour survival and anti-tumour responses.

Epstein-Barr virus LMP2A imposes sensitivity to apoptosis.

In cell lines, the Epstein-Barr virus (EBV)-encoded protein latent membrane protein 2A (LMP2A) protects B-cells from apoptosis by blocking B-cell receptor (BCR) signalling. However, EBV-infected B-cells in vivo are extremely different from cell lines. This study used a murine transgenic model in which B-cells express LMP2A and a BCR specific for hen egg lysozyme to determine whether LMP2A protects resting and antigen-activated B-cells from apoptosis. LMP2A allows BCR signal transduction and induces constitutive activation of NF-kappaB to increase Bcl-2 levels that afford LMP2A-mediated protection from apoptosis in the absence or presence of antigen. In contrast, low levels of NF-kappaB inhibitor only affected Bcl-2 and Bcl-xL levels and increased apoptosis in LMP2A-negative B-cells after BCR cross-linking. These data suggest that LMP2A uniquely makes resting B-cells sensitive to NF-kappaB inhibition and apoptosis and suggest that NF-kappaB may be a novel target to eradicate latently EBV-infected B-cells.

Epstein-Barr virus in Burkitt's lymphoma: a role for latent membrane protein 2A.

Burkitt's lymphoma (BL) is characterized by translocation of the MYC gene to an immunoglobulin locus. Transgenic mouse models have been used to study the molecular changes that are necessary to bypass tumor suppression in the presence of translocated MYC. Inactivation of the p53 pathway is a major step to tumor formation in mouse models that is also seen in human disease. Human BL is often highly associated with Epstein-Barr virus (EBV). The EBV latency protein latent membrane protein 2A (LMP2A) is known to promote B cell survival by affecting levels of pro-survival factors. Using LMP2A transgenic mouse models, we have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work uncovers a contribution of EBV to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL.

Epstein-Barr virus latent membrane protein 2A and autoimmunity.

Epstein-Barr virus (EBV) has been associated with autoimmune diseases for over 40 years. However, the mechanisms by which EBV might promote autoimmune development remain elusive. Many of the hypotheses for the means by which EBV might achieve this incorporate the idea that autoimmune responses are initially immune responses against EBV proteins that crossreact with endogenous human proteins. However, recent evidence using transgenic mouse models suggests that B cells expressing the EBV-encoded protein latent membrane protein 2A (LMP2A) bypasses normal tolerance checkpoints and enhances the development of autoimmune diseases. Evidence from transgenic mouse models supports a paradigm in which LMP2A could promote autoimmune development. This novel model provides a framework to test potential mechanisms by which EBV could promote the development of autoimmune responses and might enable the identification of strategies to treat EBV-associated autoimmune diseases.

Epstein-Barr virus LMP2A enhances B-cell responses in vivo and in vitro.

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.

Epstein-Barr virus LMP2A alters in vivo and in vitro models of B-cell anergy, but not deletion, in response to autoantigen.

A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-kappaB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-kappaB nuclear translocation independent of BCR cross-linking. Since NF-kappaB is required to bypass tolerance induction, this LMP2A-dependent NF-kappaB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.

Adaptive immunity in mice lacking the beta(2)-adrenergic receptor.

The beta-2-adrenergic receptor (beta(2)AR) is expressed by most lymphocyte populations and binds the sympathetic neurotransmitter norepinephrine (NE). Stimulation of the beta(2)AR is reported to be the primary mechanism by which signals from the sympathetic nervous system influence both cell-mediated and humoral immunity. We report here that body/organ weights, lymphoid organ cell number/phenotype/histology, the contact sensitivity response, and the amount, avidity, and isotype of antibody resulting from a T cell-dependent antibody response in beta(2)AR deficient mice (beta(2)AR-/- mice) were all similar to measures made in beta(2)AR+/+ mice. Other members of the adrenergic receptor family did not appear to compensate for the absence in beta(2)AR expression. In contrast, beta(2)AR-/- B cells cultured in vitro were unable to respond to NE in a manner similar to beta(2)AR+/+ B cells. Thus, mice in which expression of the beta(2)AR gene is defective from early development to adulthood may no longer require that NE stimulate the beta(2)AR to maintain immune homeostasis, and this may be due to a non-adrenergic mechanism that provides compensation in vivo.