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.

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 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 Blockade of SARS-CoV-2 spike protein-mediated cell–cell fusion using COVID-19 convalescent plasma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ling Wang ◽  
Juan Zhao ◽  
Lam N. T. Nguyen ◽  
James L. Adkins ◽  
Madison Schank ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Neutralizing Antibodies ◽  
Molecular Mechanisms ◽  
Virus Entry ◽  
High Capacity ◽  
Target Cells ◽  
Global Public Health ◽  
S Protein ◽  
Cell Cell

AbstractThe recent COVID-19 pandemic poses a serious threat to global public health, thus there is an urgent need to define the molecular mechanisms involved in SARS-CoV-2 spike (S) protein-mediated virus entry that is essential for preventing and/or treating this emerging infectious disease. In this study, we examined the blocking activity of human COVID-19 convalescent plasma by cell–cell fusion assays using SARS-CoV-2-S-transfected 293 T as effector cells and ACE2-expressing 293 T as target cells. We demonstrate that the SARS-CoV-2 S protein exhibits a very high capacity for membrane fusion and is efficient in mediating virus fusion and entry into target cells. Importantly, we find that COVID-19 convalescent plasma with high titers of IgG neutralizing antibodies can block cell–cell fusion and virus entry by interfering with the SARS-CoV-2-S/ACE2 or SARS-CoV-S/ACE2 interactions. These findings suggest that COVID-19 convalescent plasma may not only inhibit SARS-CoV-2-S but also cross-neutralize SARS-CoV-S-mediated membrane fusion and virus entry, supporting its potential as a preventive and/or therapeutic agent against SARS-CoV-2 as well as other SARS-CoV infections.

scholarly journals Engineered Disulfide Bonds in Herpes Simplex Virus Type 1 gD Separate Receptor Binding from Fusion Initiation and Viral Entry

2007 ◽  
Vol 82 (2) ◽  
pp. 700-709 ◽  
Author(s):  
Eric Lazear ◽  
Andrea Carfi ◽  
J. Charles Whitbeck ◽  
Tina M. Cairns ◽  
Claude Krummenacher ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Disulfide Bond ◽  
Viral Entry ◽  
Disulfide Bonds ◽  
C Terminus ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT Glycoprotein D (gD) is the receptor binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, herpesvirus entry mediator (HVEM) and nectin-1. While both receptor binding regions are found within the first 234 amino acids, a crystal structure shows that the C terminus of the gD ectodomain normally occludes the receptor binding sites. Receptor binding must therefore displace the C terminus, and this conformational change is postulated to be required for inducing fusion via gB and gH/gL. When cysteine residues are introduced at positions 37 and 302 of gD, a disulfide bond is formed that stabilizes the C terminus and prevents binding to either receptor. We speculated that if disulfide bonds were engineered further upstream, receptor binding might be separated from the induction of fusion. To test this, we made five additional double cysteine mutants, each potentially introducing a disulfide bond between the ectodomain C terminus and the core of the gD ectodomain. The two mutants predicted to impose the greatest constraint were unable to bind receptors or mediate cell-cell fusion. However, the three mutants with the most flexible C terminus bound well to both HVEM and nectin-1. Two of these mutants were impaired in cell-cell fusion and null-virus complementation. Importantly, a third mutant in this group was nonfunctional in both assays. This mutant clearly separates the role of gD in triggering fusion from its role in receptor binding. Based upon the properties of the panel of mutants we conclude that fusion requires greater flexibility of the gD ectodomain C terminus than does receptor binding.

scholarly journals Using Split Luciferase Assay and anti-HSV Glycoprotein Monoclonal Antibodies to Predict a Functional Binding Site Between gD and gH/gL

2021 ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Tina M Cairns ◽  
Roselyn J Eisenberg ◽  
Gary H Cohen
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Cell Fusion ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Active Form ◽  
The Other ◽  
Luciferase Assay ◽  
Functional Sites ◽  
Cell Cell

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. HSV entry begins with gD binding its receptor (nectin-1), which then activates gH/gL to enable the conversion of pre-fusion gB to its active form to promote membrane fusion. Virus-neutralizing monoclonal antibodies (Mabs) interfere with one or more of these steps and localization of their epitopes identifies functional sites on each protein. Utilizing this approach, we have identified the gH/gL binding face on gD and the corresponding gD binding site on gH/gL. Here, we used combinations of these Mabs to define the orientation of gD and gH/gL relative to each other. We reasoned that if two Mabs, one directed at gD and the other at gH/gL, block fusion more effectively than when either were used alone (additive), then their epitopes would be spatially distanced and binding of one would not directly interfere with binding of the other during fusion. However, if the two Mabs blocked fusion with equal or lesser efficacy that when either were used alone (indifferent), we propose that their epitopes would be in close proximity in the complex. Using a live cell fusion assay, we found that some Mab pairings blocked the fusion with different mechanisms while other had a similar mechanisms of action. Grouping the different combinations of antibodies into indifferent and additive groups, we present a model for the orientation of gD vis-à-vis gH/gL in the complex. Importance: Virus entry and cell-cell fusion mediated by HSV require four essential glycoproteins, gD, gH/gL, gB and a gD receptor. Virus-neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with essential steps in the fusion pathway. Thus, the epitopes of these Mabs overlap and point to critical, functional sites on their target proteins. Here, we combined gD and gH/gL antibodies to determine whether they work in an additive or non-additive (indifferent) fashion to block specific events in glycoprotein-driven cell-cell fusion. Identifying combinations of antibodies that have additive effects will help in the rational design of an effective therapeutic “polyclonal antibody” to treat HSV disease. In addition, identification of the exact contact regions between gD and gH/gL can inform the design of small molecules that would interfere with the gD-gH/gL complex formation, thus preventing the virus from entering the host cell.

scholarly journals The Impact on Infectivity and Neutralization Efficiency of SARS-CoV-2 Lineage B.1.351 Pseudovirus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 633
Author(s):  
Yeong Jun Kim ◽  
Ui Soon Jang ◽  
Sandrine M. Soh ◽  
Joo-Youn Lee ◽  
Hye-Ra Lee
Keyword(s):  
South Africa ◽  
Monoclonal Antibodies ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Receptor Binding Domain ◽  
Viral Spread ◽  
New Variant ◽  
Global Concern ◽  
The Impact

A new variant of SARS-CoV-2 B.1.351 lineage (first found in South Africa) has been raising global concern due to its harboring of multiple mutations in the spike that potentially increase transmissibility and yield resistance to neutralizing antibodies. We here tested infectivity and neutralization efficiency of SARS-CoV-2 spike pseudoviruses bearing particular mutations of the receptor-binding domain (RBD) derived either from the Wuhan strains (referred to as D614G or with other sites) or the B.1.351 lineage (referred to as N501Y, K417N, and E484K). The three different pseudoviruses B.1.351 lineage related significantly increased infectivity compared with other mutants that indicated Wuhan strains. Interestingly, K417N and E484K mutations dramatically enhanced cell–cell fusion than N501Y even though their infectivity were similar, suggesting that K417N and E484K mutations harboring SARS-CoV-2 variant might be more transmissible than N501Y mutation containing SARS-CoV-2 variant. We also investigated the efficacy of two different monoclonal antibodies, Casirivimab and Imdevimab that neutralized SARS-CoV-2, against several kinds of pseudoviruses which indicated Wuhan or B.1.351 lineage. Remarkably, Imdevimab effectively neutralized B.1.351 lineage pseudoviruses containing N501Y, K417N, and E484K mutations, while Casirivimab partially affected them. Overall, our results underscore the importance of B.1.351 lineage SARS-CoV-2 in the viral spread and its implication for antibody efficacy.

Herpes simplex virus glycoprotein B (gB) mutations define structural sites in domain I, the membrane proximal region, and the cytodomain that regulate entry.

2021 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Cell Fusion ◽  
Virus Entry ◽  
Proximal Region ◽  
Glycoprotein B ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Membrane Proximal Region ◽  
Domain V ◽  
Domain I ◽  
Cell Cell

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

scholarly journals Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

2020 ◽  
Author(s):  
Alexandra C. Walls ◽  
Young-Jun Park ◽  
M. Alexandra Tortorici ◽  
Abigail Wall ◽  
Andrew T. McGuire ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Furin Cleavage ◽  
Humoral Immune ◽  
Binding Domains ◽  
Furin Cleavage Site ◽  
Recent Emergence ◽  
Novel Coronavirus

SUMMARYThe recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

scholarly journals The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

2003 ◽  
Vol 77 (5) ◽  
pp. 3058-3066 ◽  
Author(s):  
Laurie J. Earp ◽  
Sue E. Delos ◽  
Robert C. Netter ◽  
Paul Bates ◽  
Judith M. White
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Low Ph ◽  
Target Cells ◽  
Dependent Manner ◽  
Neutral Ph ◽  
Subtype A ◽  
Avian Sarcoma ◽  
Virus Subtype ◽  
Cell Cell

ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

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