Nipah Virus Entry and Egress from Polarized Epithelial Cells

Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal.     

Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation,Mobility, and Function to Block Syncytia Formation

Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell.     

Mutations in the Stalk Region of the Measles Virus Hemagglutinin Inhibit Syncytium Formation but Not Virus Entry

Measles virus (MV) entry requires at least 2 viral proteins, the hemagglutinin (H) and fusion (F) proteins. We describe the rescue and characterization of a measles virus with a specific mutation in the stalk region of H (I98A) that is able to bind normally to cells but infects at a lower rate than the wild type due to a reduction in fusion triggering. The mutant H protein binds to F more avidly than the parent H protein does, and the corresponding virus is more sensitive to inhibition by fusion-inhibitory peptide. We show that after binding of MV to its receptor, H-F dissociation is required for productive infection.Measles virus (MV) infection requires binding of the hemagglutinin (H) protein to its cognate receptors (9, 20, 21, 29, 41) while the fusion (F) protein triggers membrane lipid mixing and fusion. The H protein is a type II transmembrane homodimeric, disulfide-linked glycoprotein (33). The F protein is a type I membrane glycoprotein that exists as a homotrimeric complex. The protein is cleaved by furin in the trans-Golgi network into a metastable heterodimer with a membrane-spanning F₁ domain and a membrane-distal F₂ domain (16). Expressed alone, neither H nor F leads to membrane fusion, and therefore, both proteins are required and have to interact for productive infection of a target cell (46). There is evidence that these interactions start within the endoplasmic reticulum (34).The H proteins of Paramyxoviridae family members have a globular head with a six-blade β-propellor structure that is responsible for receptor binding (4, 7, 13), a stalk region composed of alpha-helical coiled coils (18, 48) that anchors the complex to the plasma membrane, and a short cytoplasmic domain that can interact with the matrix (M) protein and modulate fusion (2). Given that the F protein does not interact with a receptor on the target cell but undergoes conformational changes to enable membrane fusion, it seems likely that the F protein must interact with the H protein that enables fusion (14, 19, 23, 24, 35, 47). The molecular interactions between the F and H proteins are being increasingly understood (6, 8, 24, 25, 30, 35, 42). Hummel and Bellini have described a mutation in the H glycoprotein where threonine replaced isoleucine 98, which led to loss of fusion in chronically infected cells, but the virus was not rescued (15). Corey and Iorio performed alanine-scanning mutagenesis to determine the role of specific, membrane-proximal residues in the stalk region of the H protein responsible for H-F interactions (6). Substitution of alanine for specific residues in this region altered cell-to-cell fusion and the strength of the H-F interaction in transient-transfection experiments (6). Replacement of isoleucine with alanine at position 98 reduced fusion but did not significantly alter hemadsorption, implying that binding of the mutant H protein to CD46 was not affected (6). More recently, Paal et al. showed that the H protein can tolerate significant additions to its alpha-helical coiled coils without loss of binding or fusion in transient-transfection assays (30). Although these studies confirm the importance of the interactions between the H protein stalk and the metastable F protein for enabling fusion after receptor binding, the exact steps leading to fusion are still unclear. Moreover, studies evaluating H-F interactions were performed with transient protein expression and not in the presence of the actual virus. This is potentially an important shortcoming since the M protein can modulate infection and fusion (1).     

Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone.     

Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding.     

Fluid Flow Modulates Calcium Entry and Activates Membrane Currents in Cultured Human Aortic Endothelial Cells

Human aortic endothelial cells (HAEC) respond to flow with Ca²⁺ entry, activation of a nonselective cation channel, activation of a chloride channel, and activation of a calcium-activated potassium channel. Conversely, human capillary endothelial cells were unaffected by similar flow rates. In HAEC the flow induced cytosolic free calcium increase ([Ca²⁺] _i ) and the ionic currents associated with it were sustained for up to 15 min after perfusion was stopped. In the absence of extracellular Ca²⁺, fluid flow was unable to evoke the [Ca²⁺] _i increase or the increase in membrane currents but the response could be restored by addition of extracellular Ca²⁺. Surprisingly, the flow response was inhibited in 50% of the cells by inhibitors of nitric oxide production. The results suggest that the sustained flow response in HAEC may be partially mediated by nitric oxide production and release. Received: 29 January 1999/Revised: 2 June 1999     

Rapid Fusion and Syncytium Formation of Heterologous Cells upon Expression of the FGFRL1 Receptor

The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.     

Expression of Thrombospondin-1 Modulates the Angioinflammatory Phenotype of Choroidal Endothelial Cells

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1−/−) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1−/− ChEC migration and capillary morphogenesis. TSP1−/− ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1−/− ChEC produced similar levels of VEGF, although TSP1−/− ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.     

Inhibition of pRB Pathway Differentially Modulates Apoptosis in Esophageal Cancer Cells

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Current chemotherapy regimens include a combination of 5-fluorouracil (5-FU) and cisplatin, but more efficient therapy strategies are needed to increase 5-year survival. Alterations in the signaling pathway of the tumor suppressor gene Rb-1, which encodes a phosphoprotein (pRB) that negatively regulates the G1/S transition of the cell cycle, are present in 70% of all tumors, but its role in esophageal cancer is still unclear. Most of these are alterations leading to up-regulation of the activity of cyclin-dependent kinases (CDKs) to phosphorylate pRB, which suggests that keeping the wild type pRB phosphorylated might be advantageous. Besides proliferation, pRB also regulates apoptosis induced by tumor necrosis factor-alpha (TNF-α) and DNA-damage. We investigated the status of phosphorylation of pRB along esophageal tumorigenesis stages, as well as whether hyperphosphorylation of pRB could suppress apoptosis induced by cisplatin, 5-FU, or TNF-α in esophageal cancer cells. pRB phosphorylation increased progressively from normal esophageal tissue to metaplasia and adenocarcinoma, suggesting that pRB phosphorylation increases along esophageal tumor stages. When RB-1 was knocked down or CDK inhibitors reduced the levels of phosphorylated pRB, opposite apoptotic effects were observed, depending on the combination of drugs tested: whereas TNF-α- and cisplatin-induced apoptosis increased, 5-FU-induced apoptosis decreased. Taken together, these data suggest that pRB plays a role in esophageal adenocarcinoma and that, depending on the type of anti-cancer treatment, combining CDK inhibitors and chemotherapy has the potential to increase the sensitivity of esophageal cancer cells to cell death.     

Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.     

Proteolytic Activation of the Spike Protein at a Novel RRRR/S Motif Is Implicated in Furin-Dependent Entry,Syncytium Formation,and Infectivity of Coronavirus Infectious Bronchitis Virus in Cultured Cells

The spike (S) protein of the coronavirus (CoV) infectious bronchitis virus (IBV) is cleaved into S1 and S2 subunits at the furin consensus motif RRFRR₅₃₇/S in virus-infected cells. In this study, we observe that the S2 subunit of the IBV Beaudette strain is additionally cleaved at the second furin site (RRRR₆₉₀/S) in cells expressing S constructs and in virus-infected cells. Detailed time course experiments showed that a peptide furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, blocked both viral entry and syncytium formation. Site-directed mutagenesis studies revealed that the S1/S2 cleavage by furin was not necessary for, but could promote, syncytium formation by and infectivity of IBV in Vero cells. In contrast, the second site is involved in the furin dependence of viral entry and syncytium formation. Mutations of the second site from furin-cleavable RRRR/S to non-furin-cleavable PRRRS and AAARS, respectively, abrogated the furin dependence of IBV entry. Instead, a yet-to-be-identified serine protease(s) was involved, as revealed by protease inhibitor studies. Furthermore, sequence analysis of CoV S proteins by multiple alignments showed conservation of an XXXR/S motif, cleavable by either furin or other trypsin-like proteases, at a position equivalent to the second IBV furin site. Taken together, these results suggest that proteolysis at a novel XXXR/S motif in the S2 subunit might be a common mechanism for the entry of CoV into cells.The surface glycoproteins of numerous pathogenic enveloped viruses are proteolytically matured during infection in the host or cultured cell lines and are essential for the initiation of infection (33). In many cases, this processing is carried out by cellular proprotein convertases (PCs), most commonly furin (reviewed in reference 46). Furin is a calcium-dependent serine protease that circulates between the trans-Golgi network, plasma membrane, and early endosome by association with exocytic and endocytic pathways (9, 39). This membrane-bound enzyme undergoes further processing and is secreted from cells in an active soluble form (49). Furin processes a wide variety of precursor proteins after the C-terminal arginine (R) residue in the preferred consensus motif RXR(K)R/X (K is lysine, X is any amino acid, and the slash [/] indicates the cleavage position) for viral fusion proteins (2, 32, 33). So far, seven PCs have been identified in mammalian cells, and they display similar, but not identical, specificities for basic motifs at the cleavage site of a substrate. Accumulated studies indicate that secretory PCs, such as furin, PC5, and PC7, are major candidates for processing surface glycoproteins of pathogenic viruses, such as human immunodeficiency virus types 1 and 2, avian influenza virus H5N1, Ebola virus, and respiratory syncytial virus (RSV) (2, 27).Coronavirus (CoV) spike (S) protein, a class I viral fusion protein (7), is responsible for viral attachment to and entry into target cells and for cell-to-cell spread during infection. Typical class I fusion proteins usually require processing at a position immediately upstream of the fusion peptide in order to expose the membrane-anchored subunit. However, in infectious bronchitis virus (IBV) and murine hepatitis virus (MHV), processing of the S protein by furin occurs at a position more than 200 amino acids away from the predicted fusion peptides (6). Furthermore, there is a tradeoff between the furin cleavability of S protein and heparin sulfate (HS) binding in certain CoV strains adapted to cultured cell lines (15, 17). Consequently, CoV S proteins may be proteolytically activated by other proteases to initiate virus-cell fusion. Recently, proteolytic activation by an endosomal protease, cathepsin L, and a membrane-bound protease, factor Xa, was reported to play a role in the entry of severe acute respiratory syndrome (SARS)-CoV (18, 45). Cathepsin is also implicated in the proteolytic activation of many CoV S proteins, including human CoV 229E, feline infectious peritonitis virus (FIPV) 1146, feline enteric CoV (FECV) 1683, and MHV strain 2 (MHV-2), but not for MHV A59 and human CoV NL63 (31, 41, 43, 45).The association of cell surface sialic acid and a low-pH environment were reported to be required for IBV entry (14, 51, 52). However, the factors that determine the infectivity of IBV for cultured cells have yet to be identified. Clinical and field isolates of IBV can be propagated only in embryonated chicken eggs or, transiently, in primary chicken embryo kidney cells. In contrast, IBV of Beaudette strain origin can be readily adapted to cultured cells, such as Vero and BHK-21, by serial passages (1, 22, 40), and hence, it is often used as an in vitro infection model of IBV. Studies with a recombinant infectious clone system demonstrated that IBV S protein is indeed the determinant of extended cell tropism (12). IBV S protein is usually cleaved into S1 and S2 subunits at the furin consensus motif, RRFRR₅₃₇/S (the position includes the signal peptide) in virus-infected cells (13). Interestingly, Beaudette and related strains carry a mutation at position 687 of the S protein from proline (P) to R, creating a novel furin site (RRRR₆₉₀/S or RRKR₆₉₀/S). The acquisition of an additional furin site in the fusion protein may increase cell-to-cell spread by further activation of the protein (23) or extend the host range by utilization of cell surface HS as an entry receptor (17). In this study, furin-mediated cleavage of the IBV S protein at two furin sites was observed in IBV-infected cells. Mutational analysis of the two furin sites revealed that the second site is implicated in the furin dependence of IBV entry and syncytium formation. In contrast, cleavage at the S1/S2 site by furin was not necessary for, but could promote, syncytium formation and the infectivity of IBV in Vero cells.     

Fluid Shear Stress on Endothelial Cells Modulates Mechanical Tension across VE-Cadherin and PECAM-1

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Characterization of New Syncytium-Inhibiting Monoclonal Antibodies Implicates Lipid Rafts in Human T-Cell Leukemia Virus Type 1 Syncytium Formation

We have previously shown that erythroleukemia cells (K562) transfected with vascular adhesion molecule 1 (VCAM-1) are susceptible to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation. Since expression of VCAM-1 alone is not sufficient to render cells susceptible to HTLV-1 fusion, K562 cells appear to express a second molecule critical for HTLV-induced syncytium formation. By immunizing mice with K562 cells, we have isolated four monoclonal antibodies (MAbs), K5.M1, K5.M2, K5.M3, and K5.M4, that inhibit HTLV-induced syncytium formation between infected MT2 cells and susceptible K562/VCAM1 cells. These MAbs recognize distinct proteins on the surface of cells as determined by cell phenotyping, immunoprecipitation, and Western blot analysis. Since three of the proteins recognized by the MAbs appear to be GPI linked, we isolated lipid rafts and determined by immunoblot analysis that all four MAbs recognize proteins that sort entirely or in large part to lipid rafts. Dispersion of lipid rafts on the cells by cholesterol depletion with beta-cyclodextrin resulted in inhibition of syncytium formation, and this effect was not seen when the beta-cyclodextrin was preloaded with cholesterol before treating the cells. The results of these studies suggest that lipid rafts may play an important role in HTLV-1 syncytium formation.     

Thrombospondin-1 Modulates Angiogenesisin Vitroby Up-Regulation of Matrix Metalloproteinase-9 in Endothelial Cells

Evidence suggests that thrombospondin-1 (TSP-1), a 450-kDa glycoprotein in platelets and extracellular matrix, is involved in angiogenesis. However, the mechanisms by which TSP-1 regulates angiogenesis are unknown, and the exact role of TSP-1 in angiogenesis has been controversial: both stimulatory and inhibitory effects of TSP-1 have been reported. In this study, we evaluated the effect of TSP-1 on the capacity of bovine aortic endothelial (BAE) cells to both invade and form microvessel-like tubes in collagen gels. BAE cell tube formation was enhanced by exogenous TSP-1 at relatively low concentrations (1–10 μg/ml) but inhibited at higher concentrations of TSP-1 (>15 μg/ml). In addition, we correlated this biphasic effect on tube formation with the capacity of TSP-1 to stimulate the activity of a matrix metalloproteinase-9 (MMP-9) in BAE cell collagen gel cultures. The TSP-1-mediated stimulation of MMP-9 activity was specific and dose- and time-dependent. Furthermore, TSP-1-stimulated BAE cell invasion and tube formation were reversed by antibodies against both TSP-1 and MMP-9, suggesting that TSP-1 modulates endothelial cell invasion and morphogenesisin vitroby a mechanism involving the regulation of MMP-9 activity. These findings support the conclusion that TSP-1 is a multifunctional modulator of angiogenesis and are consistent with the dynamic presence of TSP-1 in remodeling tissues in which matrix degradation is required.     

A Controlled Trial to Reduce the Risk of Human Nipah Virus Exposure in Bangladesh

Human Nipah virus (NiV) infection, often fatal in Bangladesh, is primarily transmitted by drinking raw date palm sap contaminated by Pteropus bats. We assessed the impact of a behavior change communication intervention on reducing consumption of potentially NiV-contaminated raw sap. During the 2012–2014 sap harvesting seasons, we implemented interventions in two areas and compared results with a control area. In one area, we disseminated a “do not drink raw sap” message and, in the other area, encouraged only drinking sap if it had been protected from bat contamination by a barrier (“only safe sap”). Post-intervention, 40% more respondents in both intervention areas reported knowing about a disease contracted through raw sap consumption compared with control. Reported raw sap consumption decreased in all areas. The reductions in the intervention areas were not significantly greater compared to the control. Respondents directly exposed to the “only safe sap” message were more likely to report consuming raw sap from a protected source than those with no exposure (25 vs. 15%, OR 2.0, 95% CI 1.5–2.6, P < 0.001). While the intervention increased knowledge in both intervention areas, the “only safe sap” intervention reduced exposure to potentially NiV-contaminated sap and should be considered for future dissemination.     

RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.