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1.
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.  相似文献   

2.
Nipah virus (NiV) and Hendra virus (HeV) are novel paramyxoviruses from pigs and horses, respectively, that are responsible for fatal zoonotic infections of humans. The unique genetic and biological characteristics of these emerging agents has led to their classification as the prototypic members of a new genus within the Paramyxovirinae subfamily called HENIPAVIRUS: These viruses are most closely related to members of the genus Morbillivirus and infect cells through a pH-independent membrane fusion event mediated by the actions of their attachment (G) and fusion (F) glycoproteins. Understanding their cell biological features and exploring the functional characteristics of the NiV and HeV glycoproteins will help define important properties of these emerging viruses and may provide new insights into paramyxovirus membrane fusion mechanisms. Using a recombinant vaccinia virus system and a quantitative assay for fusion, we demonstrate NiV glycoprotein function and the same pattern of cellular tropism recently reported for HeV-mediated fusion, suggesting that NiV likely uses the same cellular receptor for infection. Fusion specificity was verified by inhibition with a specific antiserum or peptides derived from the alpha-helical heptads of NiV or HeV F. Like that of HeV, NiV-mediated fusion also requires both F and G. Finally, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but here we show that the NiV and HeV glycoproteins are capable of highly efficient heterotypic functional activity with each other. However, no heterotypic activity was observed with envelope glycoproteins of the morbilliviruses Measles virus and Canine distemper virus.  相似文献   

3.
Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.  相似文献   

4.
Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry.  相似文献   

5.
Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.  相似文献   

6.
Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae, which are distinguished by their ability to cause fatal disease in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) glycoproteins. Previously, we reported on HeV- and NiV-mediated fusion activities and detailed their host-cell tropism characteristics. These studies also suggested that a common cell surface receptor, which could be destroyed by protease, was utilized by both viruses. To further characterize the G glycoprotein and its unknown receptor, soluble forms of HeV G (sG) were constructed by replacing its cytoplasmic tail and transmembrane domains with an immunoglobulin kappa leader sequence coupled to either an S-peptide tag (sG(S-tag)) or myc-epitope tag (sG(myc-tag)) to facilitate purification and detection. Expression of sG was verified in cell lysates and culture supernatants by specific affinity precipitation. Analysis of sG by size exclusion chromatography and sucrose gradient centrifugation demonstrated tetrameric, dimeric, and monomeric species, with the majority of the sG released as a disulfide-linked dimer. Immunofluorescence staining revealed that sG specifically bound to HeV and NiV infection-permissive cells but not to a nonpermissive HeLa cell line clone, suggesting that it binds to virus receptor on host cells. Preincubation of host cells with sG resulted in dose-dependent inhibition of both HeV and NiV cell fusion as well as infection by live virus. Taken together, these data indicate that sG retains important native structural features, and we further demonstrate that administration of sG to rabbits can elicit a potent cross-reactive neutralizing antibody response against infectious HeV and NiV. This HeV sG glycoprotein will be exceedingly useful for structural studies, receptor identification strategies, and vaccine development goals for these important emerging viral agents.  相似文献   

7.
Dendritic cells (DC) are professional APC that control the balance between T cell immunity and tolerance. Genetic engineering of DC to regulate the outcome of the immune response is an area of intense research. Galectin (gal)-1 is an endogenous lectin that binds to glycoproteins and exerts potent regulatory effects on T cells. Consequently, gal-1 participates in central deletion of thymocytes and exerts therapeutic effects on experimental models of T cell-mediated autoimmune disorders and graft-vs-host disease. Together, these observations strongly indicate that engineering DC to express transgenic (tg) gal-1 may be beneficial to treat T cell-mediated disorders. In this study, we have investigated the impact of the expression of high levels of tg gal-1 on maturation/activation of DC and on their T cell stimulatory function. Murine DC were transduced with a recombinant adenovirus encoding hu gal-1 (gal-1-DC). Tg gal-1 was exported by a nonclassical pathway through exosomes and was retained on the DC surface inducing segregation of its ligand CD43. Expression of tg gal-1 triggered activation of DC determined by induction of a more mature phenotype, increased levels of mRNA for proinflammatory cytokines, and enhanced ability to stimulate naive T cells. Conversely, gal-1-DC induced rapid apoptosis of activated T cells. In vivo, gal-1-DC increased significantly the sensitization phase of contact hypersensitivity assays while inducing a drastic inhibition of the elicitation phase by triggering apoptosis of activated T cells in the dermis. Gal-1-DC represent a novel tool to control differentially the afferent and efferent arms of the T cell response.  相似文献   

8.
The entry of enveloped viruses into animal cells and the cell-to-cell spread of infection via cell fusion require the membrane-fusing activity of viral glycoproteins. This activity can be dependent on variable cell factors or triggered by environmental factors. Here we show that cell fusion induced by herpes simplex virus glycoproteins is dependent on the presence of cell surface glycosaminoglycans, principally heparan sulfate, or on the addition of heparin to the medium. The role of the glycosaminoglycan is probably to alter the conformation of a viral heparin-binding glycoprotein required for the fusion.  相似文献   

9.
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.  相似文献   

10.
Henipavirus is a new genus of Paramyxoviridae that uses protein-based receptors (ephrinB2 and ephrinB3) for virus entry. Paramyxovirus entry requires the coordinated action of the fusion (F) and attachment viral envelope glycoproteins. Receptor binding to the attachment protein triggers F to undergo a conformational cascade that results in membrane fusion. The accumulation of structural and functional studies on many paramyxoviral fusion and attachment proteins, including the recent elucidation of structures of Nipah virus (NiV) and Hendra virus (HeV) G glycoproteins bound and unbound to cognate ephrinB receptors, indicate that henipavirus entry and fusion could differ mechanistically from paramyxoviruses that use glycan-based receptors.  相似文献   

11.
Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.  相似文献   

12.
J Cao  I W Park  A Cooper    J Sodroski 《Journal of virology》1996,70(3):1340-1354
Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single-cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1.  相似文献   

13.
Members of the ephrin cell-surface protein family interact with the Eph receptors, the largest family of receptor tyrosine kinases, mediating bi-directional signaling during tumorogenesis and various developmental events. Surprisingly, ephrin-B2 and -B3 were recently identified as entry receptors for henipaviruses, emerging zoonotic paramyxoviruses responsible for repeated outbreaks in humans and animals in Australia, Southeast Asia, India and Bangladesh. Nipah virus (NiV) and Hendra virus (HeV) are the only two identified members in the henipavirus genus. While the initial human infection cases came from contact with infected pigs (NiV) or horses (HeV), in the more recent outbreaks of NiV both food-borne and human-to-human transmission were reported. These characteristics, together with high mortality and morbidity rates and lack of effective anti-viral therapies, make the henipaviruses a potential biological-agent threat. Viral entry is an important target for the development of anti-viral drugs. The entry of henipavirus is initiated by the attachment of the viral G envelope glycoprotein to the host cell receptors ephrin-B2 and/or -B3, followed by activation of the F fusion protein, which triggers fusion between the viral envelop and the host membrane. We review recent progress in the study of henipavirus entry, particularly the identification of ephrins as their entry receptors, and the structural characterization of the ephrin/Henipa-G interactions.  相似文献   

14.
Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large na?ve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 microg/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 microg/ml, and 98% neutralization required only 1.6 microg/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.  相似文献   

15.
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.  相似文献   

16.
Despite of differences in replication strategy among virus families, some basic principles have remained similar. Analogous mechanisms govern virus entry into cells and the use of enzymes which direct the replication of the virus genome. The function of many cell surface receptors (such as glycosoaminoglycans, glycoproteins, proteins) which interact with viral capsid proteins or envelope glycoproteins has recently been elucidated. The list of cellular receptors (Table I) is still far from being final. The capsid components, similarly as the envelope glycoproteins, may form specific pocket like sites, which interact with the cell surface receptors. Neutralizing antibodies usually react with antigenic domains adjacent to the receptor binding site(s) and hamper the close contact inevitable for virion attachment. In the case of more complex viruses, such as herpes simplex virus, different viral glycoproteins interact with several cellular receptors. At progressed phase of adsorption the virions are engulfed into endocytic vesicles and the virion fusion domain(s) become(s) activated. The outer capsid components of reoviruses which participate in adsorption and fusion may get activated already in the lumen of digestive tract, i.e. before their engulfment by resorptive epithelium cells. Activation of the hydrophobic fusion domain(s) is a further important step allowing to pass through the lipid bilayer when penetrating the cell membrane in order to reach the cytosol. Activation of the virion fusion domain is accomplished by a conformation change, which occurs at acid pH (influenza virus hemagglutinin, sigma 1 protein of the reovirus particle) and/or after protease treatment. The herpes simplex virus fusion factors (gD and gH) undergo conformation changes by a pH-independent mechanism triggered due to interaction with the cell surface receptor(s) and mediated by mutual interactions with the viral envelope glycoproteins. The virion capsid or envelope components participating in the entry and membrane fusion are not the only tools of virulence. The correct function of virus coded proteins, which participate in replication of the viral genome, and/or in the supply of necessary nucleotides, may be very essential. In the case of enteroviruses, which RNA interacts with ribosomes directly, the correct configuration of the non-coding viral RNA sequence is crucial for initiation of translation occurring in the absence of the classical "cap" structure.  相似文献   

17.
Peptides derived from the N- (N-HR) and C- (C-HR) terminal heptad repeat regions adjacent to the fusion peptide and transmembrane domains, respectively, of human immunodeficiency virus (HIV)-1 gp41 inhibit HIV-1 viral envelope glycoproteins (Env)-mediated cell fusion specifically. The mechanism of HIV-1 Env-mediated cell fusion and its inhibition by agents that target the N- and C-HR regions was investigated. Priming experiments with Env-expressing cells indicate that the N-HR region but not the C-HR region is exposed by treatment with sCD4 at 31 degrees C, whereas both the N- and C-HR regions are exposed at 37 degrees C.  相似文献   

18.
尼帕病毒F糖蛋白在重组牛痘病毒中的表达及鉴定   总被引:3,自引:0,他引:3  
尼帕病毒(NiV)F蛋白在病毒侵入细胞和诱导中和抗体等方面具有重要作用。通过over-lapping PCR合成密码子优化的F蛋白基因构建了表达NiV F蛋白的重组牛痘病毒(WR株)rWR-NiV-F。利用兔抗NiV血清为检测抗体,通过间接免疫荧光(IFA)检测到了F蛋白在重组病毒感染细胞中的表达。SDS-PAGE和Western blot检测证明重组蛋白F0被裂解为F1和F2。以rWR-NiV-F感染瞬时转染共表达NiV受体结合囊膜糖蛋白G的BHK细胞,可诱导细胞膜融合及合包体形成,证明该重组病毒表达F蛋白保持良好的抗原性及生物学活性,为NiV诊断及重组活载体疫苗研究奠定了重要基础。  相似文献   

19.
T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.  相似文献   

20.
Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates exclusively in the cytoplasm of infected cells. The viral envelope glycoproteins, E1 and E2, appear to be retained in the endoplasmic reticulum, where viral budding is thought to occur. Surprisingly, we found that the expression system used to generate HCV envelope glycoproteins influences their subcellular localization and processing. These findings have important implications for optimizing novel HCV fusion and entry assays as well as for budding and virus particle formation.  相似文献   

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