Identification of Hendra virus G glycoprotein residues that are critical for receptor binding

Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.     

Modes of paramyxovirus fusion: a Henipavirus perspective

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.     

The Mechanism of Henipavirus Fusion： Examining the Relationships between the Attachment and Fusion Glycoproteins

The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.     

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

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.     

Individual N-Glycans Added at Intervals along the Stalk of the Nipah Virus G Protein Prevent Fusion but Do Not Block the Interaction with the Homologous F Protein

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven introduced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the ability of the G protein to complement the homologous F protein in the promotion of fusion. The most membrane-proximal added N-glycan reduces fusion by 80%. However, unlike similar NDV HN and MV H mutants, the NiV G glycosylation stalk mutants retain the ability to bind F, indicating that the fusion deficiency of these mutants is not due to prevention of the G-F interaction. These findings suggest that the G-F interaction is not mediated entirely by the stalk domain of G and may be more complex than that of HN/H-F.     

Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.     

A Novel Receptor-induced Activation Site in the Nipah Virus Attachment Glycoprotein (G) Involved in Triggering the Fusion Glycoprotein (F)

Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation site in Nipah virus (NiV) G involved in triggering F-mediated fusion. We first generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (β6S4/β1H1). Alanine scan mutants within this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with F at wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry.The paramyxoviruses comprise a group of important human pathogens, such as measles, mumps, human parainfluenza viruses, and the highly pathogenic Nipah (NiV)⁴ and Hendra (HeV) viruses. NiV infections have a mortality rate in humans of up to 75%, and NiV is classified as a BSL4 pathogen because of its bio- or agro-terrorism potential (1). The efficacy of entry inhibitors targeted against HIV suggests that a better understanding of Paramyxovirus entry and fusion will facilitate similarly efficacious antiviral therapeutics.Although past studies have identified regions in either the fusion (F) or attachment (G/H/HN) glycoproteins that are important for membrane fusion or F-G/H/HN association (2–10), the region(s) in G important for receptor-activated triggering of F-mediated fusion remains unknown. Current models of Paramyxovirus membrane fusion posit that receptor binding to the attachment glycoprotein (G, H, or HN) triggers a conformational cascade in the fusion protein (F). Such F-triggering results in fusion peptide (FP) exposure, which involves formation of a pre-hairpin intermediate and subsequent six-helix bundle formation (11). The energy released upon refolding into the stable six-helix bundle ground state is what drives the fusion of the viral and host-cell membranes. These are common functional and structural features responsible for membrane fusion for all enveloped viruses regardless of whether the fusion protein has predominantly trimeric α-helical coiled-coil (Class I), β (Class II), or a combination of α and β (Class III) core structures (12). Important human pathogens such as the HIV, influenza, and various paramyxoviruses have Class I fusion proteins, and their similar structural features point to similar membrane fusion mechanisms (11, 12). Besides sharing trimeric coiled-coil structures, they are synthesized as precursors that are cleaved into a metastable conformation; cleavage generates a new hydrophobic N terminus FP that gets released and inserted into the target cell membrane upon triggering (11, 12). Class I fusion proteins have two heptad repeat regions, HR1 and HR2, at their N and C termini, respectively, that fold up onto each other during six-helix bundle formation to bring about merging of target cell and viral membranes (12). For Paramyxovirus F proteins, the C-terminal HR2 region is generally thought to be pre-formed, but the N-terminal HR1 region is formed only upon F-triggering and FP insertion (11, 13). The formation of this trimeric HR1 core just before six-helix bundle formation, is known as the pre-hairpin intermediate.Despite their common features, viral fusion proteins vary in their detailed structures, triggering factors, and number of viral surface proteins involved. For paramyxoviruses, receptor binding and fusion functions are carried out by two distinct transmembrane proteins (attachment (G, H, or HN) and fusion (F) proteins, respectively), and with few exceptions both are required for membrane fusion. The underlying mechanism of fusion triggering by the attachment protein may vary depending on their use of protein versus carbohydrate receptors (14). For example, we and others have observed an inverse correlation between fusogenicity of the F protein and the avidity of the F/G or F/H interactions for NiV and measles virus (2, 3, 5, 15, 16), both of which use protein-based receptors; however, for Newcastle disease virus, a glycan-using Paramyxovirus, there seems to be a direct correlation between fusogenicity and the avidity of F/HN interactions (8).For the paramyxoviruses, the early steps in the fusion cascade, particularly how H/HN “triggers” F, are not well understood, and the region(s) in H/HN responsible for F triggering remains unclear, although the stalk domain of H/HN appears to be important for F triggering or for interaction with F (5–8). For NiV, the G attachment glycoprotein binds either the ephrinB2 (B2) or ephrinB3 (B3) protein receptors (17–19), but it is not known how receptor engagement induces G to undergo the allosteric changes involved in triggering F. However, by homology to H or HN, it is likely that the stalk domain in NiV-G is also involved in F-triggering (20). Here we analyze the early steps in the fusion cascade for NiV and identify a specific region in NiV-G distinct from the receptor binding site that is involved in 1) B2-induced changes that trigger FP exposure in F, 2) modulating the avidity of F/G interactions resulting in displacement of F from G, and 3) transducing receptor-induced membrane fusion. Our results offer testable hypotheses as to whether this model of fusion cascade holds true for other paramyxoviruses that use protein-based receptors.     

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.     

Cysteines in the stalk of the nipah virus G glycoprotein are located in a distinct subdomain critical for fusion activation

Paramyxoviruses initiate entry through the concerted action of the tetrameric attachment glycoprotein (HN, H, or G) and the trimeric fusion glycoprotein (F). The ectodomains of HN/H/G contain a stalk region important for oligomeric stability and for the F triggering resulting in membrane fusion. Paramyxovirus HN, H, and G form a dimer-of-dimers consisting of disulfide-linked dimers through their stalk domain cysteines. The G attachment protein stalk domain of the highly pathogenic Nipah virus (NiV) contains a distinct but uncharacterized cluster of three cysteine residues (C146, C158, C162). On the basis of a panoply of assays, we report that C158 and C162 of NiV-G likely mediate covalent subunit dimerization, while C146 mediates the stability of higher-order oligomers. For HN or H, mutation of stalk cysteines attenuates but does not abrogate the ability to trigger fusion. In contrast, the NiV-G stalk cysteine mutants were completely deficient in triggering fusion, even though they could still bind the ephrinB2 receptor and associate with F. Interestingly, all cysteine stalk mutants exhibited constitutive exposure of the Mab45 receptor binding-enhanced epitope, previously implicated in F triggering. The enhanced binding of Mab45 to the cysteine mutants relative to wild-type NiV-G, without the addition of the receptor, implicates the stalk cysteines in the stabilization of a pre-receptor-bound conformation and the regulation of F triggering. Sequence alignments revealed that the stalk cysteines were adjacent to a proline-rich microdomain unique to the Henipavirus genus. Our data propose that the cysteine cluster in the NiV-G stalk functions to maintain oligomeric stability but is more importantly involved in stabilizing a unique microdomain critical for triggering fusion.     

N-Glycans on the Nipah Virus Attachment Glycoprotein Modulate Fusion and Viral Entry as They Protect against Antibody Neutralization

Nipah virus (NiV) is the deadliest known paramyxovirus. Membrane fusion is essential for NiV entry into host cells and for the virus'' pathological induction of cell-cell fusion (syncytia). The mechanism by which the attachment glycoprotein (G), upon binding to the cell receptors ephrinB2 or ephrinB3, triggers the fusion glycoprotein (F) to execute membrane fusion is largely unknown. N-glycans on paramyxovirus glycoproteins are generally required for proper protein conformational integrity, transport, and sometimes biological functions. We made conservative mutations (Asn to Gln) at the seven potential N-glycosylation sites in the NiV G ectodomain (G1 to G7) individually or in combination. Six of the seven N-glycosylation sites were found to be glycosylated. Moreover, pseudotyped virions carrying these N-glycan mutants had increased antibody neutralization sensitivities. Interestingly, our results revealed hyperfusogenic and hypofusogenic phenotypes for mutants that bound ephrinB2 at wild-type levels, and the mutant''s cell-cell fusion phenotypes generally correlated to viral entry levels. In addition, when removing multiple N-glycans simultaneously, we observed synergistic or dominant-negative membrane fusion phenotypes. Interestingly, our data indicated that 4- to 6-fold increases in fusogenicity resulted from multiple mechanisms, including but not restricted to the increase of F triggering. Altogether, our results suggest that NiV-G N-glycans play a role in shielding virions against antibody neutralization, while modulating cell-cell fusion and viral entry via multiple mechanisms.     

Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

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.     

Inhibition of hendra virus fusion

Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions (HRN and HRC) near the fusion peptide and the transmembrane domains, respectively. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing F, after the initial triggering step, from forming the 6HB structure that is required for fusion. HeV peptides have previously been found to be effective at inhibiting HeV fusion. However, we found that a human parainfluenza virus 3 F-peptide is more effective at inhibiting HeV fusion than the comparable HeV-derived peptide.     

Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins

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.     

Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are lethal emerging paramyxoviruses. EphrinB2 and ephrinB3 have been identified as receptors for henipavirus entry. NiV and HeV share similar cellular tropisms and likely use an identical receptor set, although a quantitative comparison of receptor usage by NiV and HeV has not been reported. Here we show that (i) soluble NiV attachment protein G (sNiV-G) bound to cell surface-expressed ephrinB3 with a 30-fold higher affinity than that of sHeV-G, (ii) NiV envelope pseudotyped reporter virus (NiVpp) entered ephrinB3-expressing cells much more efficiently than did HeV pseudotyped particles (HeVpp), and (iii) NiVpp but not HeVpp entry was inhibited efficiently by soluble ephrinB3. These data underscore the finding that NiV uses ephrinB3 more efficiently than does HeV. Henipavirus G chimeric protein analysis implicated residue 507 in the G ectodomain in efficient ephrinB3 usage. Curiously, alternative versions of published HeV-G sequences show variations at residue 507 that can clearly affect ephrinB3 but not ephrinB2 usage. We further defined surrounding mutations (W504A and E505A) that diminished ephrinB3-dependent binding and viral entry without compromising ephrinB2 receptor usage and another mutation (E533Q) that abrogated both ephrinB2 and -B3 usage. Our results suggest that ephrinB2 and -B3 binding determinants on henipavirus G are distinct and dissociable. Global expression analysis showed that ephrinB3, but not ephrinB2, is expressed in the brain stem. Thus, ephrinB3-mediated viral entry and pathology may underlie the severe brain stem neuronal dysfunction seen in fatal Nipah viral encephalitis. Characterizing the determinants of ephrinB2 versus -B3 usage will further our understanding of henipavirus pathogenesis.     

Evidence of a potential receptor-binding site on the Nipah virus G protein (NiV-G): identification of globular head residues with a role in fusion promotion and their localization on an NiV-G structural model

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such "escape mutants" identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of beta-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin.     

Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors

The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding.     

Potent neutralization of Hendra and Nipah viruses by human monoclonal antibodies

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.     

New Insights into the Hendra Virus Attachment and Entry Process from Structures of the Virus G Glycoprotein and Its Complex with Ephrin-B2

Hendra virus and Nipah virus, comprising the genus Henipavirus, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. Two surface glycoproteins, the attachment (G) and fusion (F), mediate host cell entry. The crystal structures of the Hendra G glycoprotein alone and in complex with the ephrin-B2 receptor reveal that henipavirus uses Tryptophan 122 on ephrin-B2/B3 as a “latch” to facilitate the G-receptor association. Structural-based mutagenesis of residues in the Hendra G glycoprotein at the receptor binding interface document their importance for viral attachments and entry, and suggest that the stability of the Hendra-G-ephrin attachment complex does not strongly correlate with the efficiency of viral entry. In addition, our data indicates that conformational rearrangements of the G glycoprotein head domain upon receptor binding may be the trigger leading to the activation of the viral F fusion glycoprotein during virus infection.     

Nipah Virus Envelope-Pseudotyped Lentiviruses Efficiently Target ephrinB2-Positive Stem Cell Populations In Vitro and Bypass the Liver Sink When Administered In Vivo

Sophisticated retargeting systems for lentiviral vectors have been developed in recent years. Most seek to suppress the viral envelope''s natural tropism while modifying the receptor-binding domain such that its tropism is determined by the specificity of the engineered ligand-binding motif. Here we took advantage of the natural tropism of Nipah virus (NiV), whose attachment envelope glycoprotein has picomolar affinity for ephrinB2, a molecule proposed as a molecular marker of “stemness” (present on embryonic, hematopoietic, and neural stem cells) as well as being implicated in tumorigenesis of specific cancers. NiV entry requires both the fusion (F) and attachment (G) glycoproteins. Truncation of the NiV-F cytoplasmic tail (T5F) alone, combined with full-length NiV-G, resulted in optimal titers of NiV-pseudotyped particles (NiVpp) (∼10⁶ IU/ml), even without ultracentrifugation. To further enhance the infectivity of NiVpp, we engineered a hyperfusogenic NiV-F protein lacking an N-linked glycosylation site (T5FΔN3). T5FΔN3/wt G particles exhibited enhanced infectivity on less permissive cell lines and efficiently targeted ephrinB2⁺ cells even in a 1,000-fold excess of ephrinB2-negative cells, all without any loss of specificity, as entry was abrogated by soluble ephrinB2. NiVpp also transduced human embryonic, hematopoietic, and neural stem cell populations in an ephrinB2-dependent manner. Finally, intravenous administration of the luciferase reporter NiVpp-T5FΔN3/G to mice resulted in signals being detected in the spleen and lung but not in the liver. Bypassing the liver sink is a critical barrier for targeted gene therapy. The extraordinary specificity of NiV-G for ephrinB2 holds promise for targeting specific ephrinB2⁺ populations in vivo or in vitro.     

Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF_GCNt), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F₀ precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF_GCNt) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF_GCNt constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF_GCNt and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses.