scholarly journals Nipah Virus Attachment Glycoprotein Stalk C-Terminal Region Links Receptor Binding to Fusion Triggering

2014 ◽  
Vol 89 (3) ◽  
pp. 1838-1850 ◽  
Author(s):  
Qian Liu ◽  
Birgit Bradel-Tretheway ◽  
Abrrey I. Monreal ◽  
Jonel P. Saludes ◽  
Xiaonan Lu ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Viral Entry ◽  
Nipah Virus ◽  
Cell Receptor ◽  
Terminal Region ◽  
Content Type ◽  
Cell Cell

ABSTRACTMembrane fusion is essential for paramyxovirus entry into target cells and for the cell-cell fusion (syncytia) that results from many paramyxoviral infections. The concerted efforts of two membrane-integral viral proteins, the attachment (HN, H, or G) and fusion (F) glycoproteins, mediate membrane fusion. The emergent Nipah virus (NiV) is a highly pathogenic and deadly zoonotic paramyxovirus. We recently reported that upon cell receptor ephrinB2 or ephrinB3 binding, at least two conformational changes occur in the NiV-G head, followed by one in the NiV-G stalk, that subsequently result in F triggering and F execution of membrane fusion. However, the domains and residues in NiV-G that trigger F and the specific events that link receptor binding to F triggering are unknown. In the present study, we identified a NiV-G stalk C-terminal region (amino acids 159 to 163) that is important for multiple G functions, including G tetramerization, conformational integrity, G-F interactions, receptor-induced conformational changes in G, and F triggering. On the basis of these results, we propose that this NiV-G region serves as an important structural and functional linker between the NiV-G head and the rest of the stalk and is critical in propagating the F-triggering signal via specific conformational changes that open a concealed F-triggering domain(s) in the G stalk. These findings broaden our understanding of the mechanism(s) of receptor-induced paramyxovirus F triggering during viral entry and cell-cell fusion.IMPORTANCEThe emergent deadly viruses Nipah virus (NiV) and Hendra virus belong to theHenipavirusgenus in theParamyxoviridaefamily. NiV infections target endothelial cells and neurons and, in humans, result in 40 to 75% mortality rates. The broad tropism of the henipaviruses and the unavailability of therapeutics threaten the health of humans and livestock. Viral entry into host cells is the first step of henipavirus infections, which ultimately cause syncytium formation. After attaching to the host cell receptor, henipaviruses enter the target cell via direct viral-cell membrane fusion mediated by two membrane glycoproteins: the attachment protein (G) and the fusion protein (F). In this study, we identified and characterized a region in the NiV-G stalk C-terminal domain that links receptor binding to fusion triggering via several important glycoprotein functions. These findings advance our understanding of the membrane fusion-triggering mechanism(s) of the henipaviruses and the paramyxoviruses.

scholarly journals Novel roles of the N1 loop and N4 alpha helical region of the Nipah Virus Fusion glycoprotein in modulating early and late steps of the membrane fusion cascade

2021 ◽  
Author(s):  
J. Lizbeth Reyes Zamora ◽  
Victoria Ortega ◽  
Gunner P. Johnston ◽  
Jenny Li ◽  
Hector C. Aguilar
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Nipah Virus ◽  
Host Cells ◽  
Fusion Pore ◽  
Loop Region ◽  
Cell Cell

Nipah virus (NiV) is a zoonotic bat henipavirus in the family Paramyxoviridae. NiV is deadly to humans, infecting host cells by direct fusion of the viral and host-cell plasma membranes. This membrane fusion process is coordinated by the receptor-binding attachment (G) and fusion (F) glycoproteins. Upon G-receptor binding, F fuses membranes via a cascade that sequentially involves F-triggering, fusion-pore formation, and viral or genome entry into cells. Using NiV as an important paramyxoviral model, we identified two novel regions in F that modulate the membrane fusion cascade. For paramyxoviruses and other viral families with class I fusion proteins, the HR1 and HR2 regions in the fusion protein pre-fusion conformation bind to form a six-helix bundle in the post-fusion conformation. Here, structural comparisons between the F pre-fusion and post-fusion conformations revealed that a short loop region (N1) undergoes dramatic spatial reorganization, and a short alpha helix (N4) undergoes secondary structural changes. The roles of the N1 and N4 regions during the membrane fusion cascade, however, remain unknown for henipaviruses and paramyxoviruses. By performing alanine scan mutagenesis and various functional analyses, we report that specific residues within these regions alter various steps in the membrane fusion cascade. While the N1 region affects early F-triggering, the N4 region affects F-triggering, F thermostability, and extensive fusion-pore expansion during syncytia formation, also uncovering a link between F/G interactions and F-triggering. These novel mechanistic roles expand our understanding of henipaviral and paramyxoviral F triggering, viral entry, and cell-cell fusion (syncytia), a pathognomonic feature of paramyxoviral infections. IMPORTANCE Henipaviruses infect bats, agriculturally important animals, and humans, with high mortality rates approaching ∼75% in humans. Known human outbreaks have concentrated in southeast Asia and Australia. Further, about 20 new henipaviral species have been recently discovered in bats, with geographical spans in Asia, Africa and South America. The development of antiviral therapeutics requires a thorough understanding of the mechanism of viral entry into host cells. In this study, we discovered novel roles of two regions within the fusion protein of the deadly henipavirus NiV. Such roles were in allowing viral entry into host cells and cell-cell fusion, a pathological hallmark of this and other paramyxoviruses. These novel roles were in the previously undescribed N1 and N4 regions within the fusion protein, modulating early and late steps of these important process of viral infection and henipaviral disease. Notably, this knowledge may apply to other henipaviruses and more broadly to other paramyxoviruses.

scholarly journals Roles of Cholesterol in Early and Late Steps of the Nipah Virus Membrane Fusion Cascade

2021 ◽  
Author(s):  
Erik M. Contreras ◽  
Gunner P. Johnston ◽  
David W. Buchholz ◽  
Victoria Ortega ◽  
I. Abrrey Monreal ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Viral Entry ◽  
Nipah Virus ◽  
Fusion Pore ◽  
Membrane Cholesterol ◽  
Enveloped Viruses ◽  
Cell Cell

Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Though cholesterol has been associated with binding and entry of various enveloped viruses into cells, cholesterol’s exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Further, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytia formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MβCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (NiV/VSV) pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes. IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytia formation, and virus-cell membrane fusion during viral entry. Further, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.

scholarly journals Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus

2015 ◽  
Vol 89 (14) ◽  
pp. 7235-7247 ◽  
Author(s):  
Birgit G. Bradel-Tretheway ◽  
Qian Liu ◽  
Jacquelyn A. Stone ◽  
Samantha McInally ◽  
Hector C. Aguilar
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Immune Evasion ◽  
Viral Entry ◽  
Nipah Virus ◽  
Hendra Virus ◽  
Viral Attachment ◽  
Glycoprotein G ◽  
Protein Functions ◽  
Cell Cell

ABSTRACTHendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within theParamyxoviridaefamily. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses.IMPORTANCEViral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses.

scholarly journals Using Antibodies and Mutants To Localize the Presumptive gH/gL Binding Site on Herpes Simplex Virus gD

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Eric Lazear ◽  
J. Charles Whitbeck ◽  
Tina M. Cairns ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Protein Interactions ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Epitope Mapping ◽  
Virus Entry ◽  
Cellular Receptor ◽  
Functional Sites ◽  
Cell Cell

ABSTRACTHSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH, and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the prefusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two “faces” on gD: one for receptor binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (MAbs) into five competition communities. MAbs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays, we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other MAbs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL.IMPORTANCEVirus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

scholarly journals Conformational States of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Ectodomain

2006 ◽  
Vol 80 (14) ◽  
pp. 6794-6800 ◽  
Author(s):  
Fang Li ◽  
Marcelo Berardi ◽  
Wenhui Li ◽  
Michael Farzan ◽  
Philip R. Dormitzer ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Viral Entry ◽  
Protein S ◽  
Spike Protein ◽  
Fusion Peptides ◽  
Conformational States ◽  
Protease Cleavage

ABSTRACT The severe acute respiratory syndrome coronavirus enters cells through the activities of a spike protein (S) which has receptor-binding (S1) and membrane fusion (S2) regions. We have characterized four sequential states of a purified recombinant S ectodomain (S-e) comprising S1 and the ectodomain of S2. They are S-e monomers, uncleaved S-e trimers, cleaved S-e trimers, and dissociated S1 monomers and S2 trimer rosettes. Lowered pH induces an irreversible transition from flexible, L-shaped S-e monomers to clove-shaped trimers. Protease cleavage of the trimer occurs at the S1-S2 boundary; an ensuing S1 dissociation leads to a major rearrangement of the trimeric S2 and to formation of rosettes likely to represent clusters of elongated, postfusion trimers of S2 associated through their fusion peptides. The states and transitions of S suggest conformational changes that mediate viral entry into cells.

scholarly journals Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

2007 ◽  
Vol 81 (9) ◽  
pp. 4520-4532 ◽  
Author(s):  
Hector C. Aguilar ◽  
Kenneth A. Matreyek ◽  
Daniel Y. Choi ◽  
Claire Marie Filone ◽  
Sophia Young ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Fusion ◽  
Viral Entry ◽  
Nipah Virus ◽  
Cytoplasmic Tail ◽  
Wild Type ◽  
Strong Negative Correlation ◽  
Virus Fusion ◽  
Inside Out ◽  
Cell Cell

ABSTRACT The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ∼20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r 2 = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

Headless henipaviral receptor binding glycoproteins reveal key post-receptor binding contributions of the globular head and head/stalk interface in fusion promotion

2021 ◽  
Author(s):  
Yao Yu Yeo ◽  
David W. Buchholz ◽  
Amandine Gamble ◽  
Mason Jager ◽  
Hector C. Aguilar
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Emerging Viruses ◽  
Multinucleated Cells ◽  
Head Domain ◽  
Cell Cell

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

scholarly journals A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 229 ◽  
Author(s):  
María García-Murria ◽  
Neus Expósito-Domínguez ◽  
Gerard Duart ◽  
Ismael Mingarro ◽  
Luis Martinez-Gil
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
High Throughput Screening ◽  
Nipah Virus ◽  
Syncytium Formation ◽  
Viral Proteins ◽  
Viral Fusion ◽  
Multinucleated Cells ◽  
Virus Entry Inhibitors ◽  
Cell Cell

Fusion of viral and cellular membranes is a key step during the viral life cycle. Enveloped viruses trigger this process by means of specialized viral proteins expressed on their surface, the so-called viral fusion proteins. There are multiple assays to analyze the viral entry including those that focus on the cell-cell fusion induced by some viral proteins. These methods often rely on the identification of multinucleated cells (syncytium) as a result of cell membrane fusions. In this manuscript, we describe a novel methodology for the study of cell-cell fusion. Our approach, named Bimolecular Multicellular Complementation (BiMuC), provides an adjustable platform to qualitatively and quantitatively investigate the formation of a syncytium. Furthermore, we demonstrated that our procedure meets the requirements of a drug discovery approach and performed a proof of concept small molecule high-throughput screening to identify compounds that could block the entry of the emerging Nipah virus.

scholarly journals Cleavage of Rhesus Rotavirus VP4 after Arginine 247 Is Essential for Rotavirus-Like Particle-Induced Fusion from Without

1998 ◽  
Vol 72 (6) ◽  
pp. 5323-5327 ◽  
Author(s):  
Joanna M. Gilbert ◽  
Harry B. Greenberg
Keyword(s):  
Cell Fusion ◽  
Conformational Changes ◽  
Viral Entry ◽  
Recombinant Baculovirus ◽  
Syncytium Formation ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Fusion Activity ◽  
Arginine Residues ◽  
Cell Cell

ABSTRACT We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555–4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.

scholarly journals Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins

2016 ◽  
Vol 90 (23) ◽  
pp. 10762-10773 ◽  
Author(s):  
Jacquelyn A. Stone ◽  
Bhadra M. Vemulapati ◽  
Birgit Bradel-Tretheway ◽  
Hector C. Aguilar
Keyword(s):  
Membrane Protein ◽  
Membrane Fusion ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Viral Entry ◽  
Nipah Virus ◽  
Viral Envelope ◽  
Host Cells ◽  
Protein Protein Interactions ◽  
Flow Cytometric

ABSTRACTThe paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions.IMPORTANCENipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.

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