scholarly journals N-Glycans on Nipah Virus Fusion Protein Protect against Neutralization but Reduce Membrane Fusion and Viral Entry

2006 ◽  
Vol 80 (10) ◽  
pp. 4878-4889 ◽  
Author(s):  
Hector C. Aguilar ◽  
Kenneth A. Matreyek ◽  
Claire Marie Filone ◽  
Sara T. Hashimi ◽  
Ernest L. Levroney ◽  
...  
Keyword(s):  
Viral Entry ◽  
Nipah Virus ◽  
Syncytium Formation ◽  
Host Cells ◽  
Heptad Repeat ◽  
Attachment Protein ◽  
Negative Effect ◽  
Increased Sensitivity ◽  
Displacement Model ◽  
Shed Light

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

scholarly journals Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

2005 ◽  
Vol 79 (6) ◽  
pp. 3289-3296 ◽  
Author(s):  
Choong-Tat Keng ◽  
Aihua Zhang ◽  
Shuo Shen ◽  
Kuo-Ming Lip ◽  
Burtram C. Fielding ◽  
...  
Keyword(s):  
Amino Acids ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Antiviral Agents ◽  
Host Cells ◽  
Heptad Repeat ◽  
Antigenic Determinants ◽  
Amino Acid Residues ◽  
S Protein

ABSTRACT The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

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 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 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.

scholarly journals Receptor Binding and Low pH Coactivate Oncogenic Retrovirus Envelope-Mediated Fusion

2009 ◽  
Vol 83 (22) ◽  
pp. 11447-11455 ◽  
Author(s):  
Marceline Côté ◽  
Yi-Min Zheng ◽  
Shan-Lu Liu
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Underlying Mechanism ◽  
Low Ph ◽  
Host Cells ◽  
Heptad Repeat ◽  
Soluble Form ◽  
Alternate Model ◽  
Low Efficiency ◽  
Avian Sarcoma

ABSTRACT Fusion of enveloped viruses with host cells is triggered by either receptor binding or low pH but rarely requires both except for avian sarcoma leukosis virus (ASLV). We recently reported that membrane fusion mediated by an oncogenic Jaagsiekte sheep retrovirus (JSRV) envelope (Env) requires an acidic pH, yet receptor overexpression is required for this process to occur. Here we show that a soluble form of the JSRV receptor, sHyal2, promoted JSRV Env-mediated fusion at a low pH in normally fusion-negative cells and that this effect was blocked by a synthetic peptide analogous to the C-terminal heptad repeat of JSRV Env. In contrast to the receptor of ASLV, sHyal2 induced pronounced shedding of the JSRV surface subunit, as well as unstable conformational rearrangement of its transmembrane (TM) subunit, yet full activation of JSRV Env fusogenicity, associated with strong TM oligomerization, required both sHyal2 and low pH. Consistently, sHyal2 enabled transduction of nonpermissive cells by JSRV Env pseudovirions, with low efficiency, but substantially blocked viral entry into permissive cells at both binding and postbinding steps, indicating that sHyal2 prematurely activates JSRV Env-mediated fusion. Altogether, our study supports a model that receptor priming promotes fusion activation of JSRV Env at a low pH, and that the underlying mechanism is likely to be different from that of ASLV. Thus, JSRV may provide a useful alternate model for the better understanding of virus fusion and cell entry.

scholarly journals Expanded Tropism and Altered Activation of a Retroviral Glycoprotein Resistant to an Entry Inhibitor Peptide

2006 ◽  
Vol 80 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Sean M. Amberg ◽  
Robert C. Netter ◽  
Graham Simmons ◽  
Paul Bates
Keyword(s):  
Viral Entry ◽  
Terminal Segment ◽  
Heptad Repeat ◽  
Activation Threshold ◽  
Immunodeficiency Virus ◽  
Increased Sensitivity ◽  
Avian Sarcoma ◽  
Hiv 1

ABSTRACT The envelope of class I viruses can be a target for potent viral inhibitors, such as the human immunodeficiency virus type 1 (HIV-1) inhibitor enfuvirtide, which are derived from the C-terminal heptad repeat (HR2) of the transmembrane (TM) subunit. Resistance to an HR2-based peptide inhibitor of a model retrovirus, subgroup A of the Avian Sarcoma and Leukosis Virus genus (ASLV-A), was studied by examining mutants derived by viral passage in the presence of inhibitor. Variants with reduced sensitivity to inhibitor were readily selected in vitro. Sensitivity determinants were identified for 13 different isolates, all of which mapped to the TM subunit. These determinants were identified in two regions: (i) the N-terminal heptad repeat (HR1) and (ii) the N-terminal segment of TM, between the subunit cleavage site and the fusion peptide. The latter class of mutants identified a region outside of the predicted HR2-binding site that can significantly alter sensitivity to inhibitor. A subset of the HR1 mutants displayed the unanticipated ability to infect nonavian cells. This expanded tropism was associated with increased efficiency of envelope triggering by soluble receptor at low temperatures, as measured by protease sensitivity of the surface subunit (SU) of envelope. In addition, expanded tropism was linked for the most readily triggered mutants with increased sensitivity to neutralization by SU-specific antiserum. These observations depict a class of HR2 peptide-selected mutations with a reduced activation threshold, thereby allowing the utilization of alternative receptors for viral entry.

scholarly journals A Bimolecular Multicelular complementation system for the detection of syncytium formation: A new methodology for the identification of entry inhibitors

2019 ◽  
Author(s):  
María Jesús García-Murria ◽  
Neus Expósito-Domínguez ◽  
Ismael Mingarro ◽  
Luis Martinez-Gil
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
High Throughput Screening ◽  
Nipah Virus ◽  
Syncytium Formation ◽  
Viral Proteins ◽  
Viral Fusion ◽  
Entry Inhibitors ◽  
Multinucleated Cells ◽  
Cell Cell

AbstractFusion 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 Multicelular Complementation (BiMuC), provides an adjustable platform to investigate qualitatively and quantitatively 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.

SARS-CoV-2 Biology Insights, Part IV.Antibody-Dependent Enhancement (ADE) and the tricky “Herd Immunity”: narrative review (Preprint)

2020 ◽  
Author(s):  
Laura Lafon-Hughes
Keyword(s):  
Viral Infection ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Whooping Cough ◽  
Common Knowledge ◽  
Herd Immunity ◽  
Life Quality ◽  
Host Cells ◽  
System P

BACKGROUND It is common knowledge that vaccination has improved our life quality and expectancy since it succeeded in achieving almost eradication of several diseases including chickenpox (varicella), diphtheria, hepatitis A and B, measles, meningococcal, mumps, pneumococcal, polio, rotavirus, rubella, tetanus and whooping cough (pertussis) Vaccination success is based on vaccine induction of neutralizing antibodies that help fight the infection (e.g. by a virus), preventing the disease. Conversely, Antibody-dependent enhancement (ADE) of a viral infection occurs when anti-viral antibodies facilitate viral entry into host cells and enhance viral infection in these cells. ADE has been previously studied in Dengue and HIV viruses and explains why a second infection with Dengue can be lethal. As already reviewed in Part I and Part II, SARS-Cov-2 shares with HIV not only 4 sequences in the Spike protein but also the capacity to attack the immune system. OBJECTIVE As HIV presents ADE, we wondered whether this was also the case regarding SARS-CoV-2. METHODS A literature review was done through Google. RESULTS SARS-CoV-2 presents ADE. As SARS, which does not have the 4 HIV-like inserts, has the same property, ADE would not be driven by the HIV-like spike sequences. CONCLUSIONS ADE can explain the failure of herd immunity-based strategies and will also probably hamper anti-SARS-CoV-2 vaccine development. As reviewed in Part I, there fortunately are promising therapeutic strategies for COVID-19, which should be further developed. In the meantime, complementary countermeasures to protect mainly the youth from this infection are presented to be discussed in Part V Viewpoint.

scholarly journals SARS-CoV-2 Is Not Detected in the Cerebrospinal Fluid of Encephalopathic COVID-19 Patients

2020 ◽  
Vol 11 ◽  
Author(s):  
Dimitris G. Placantonakis ◽  
Maria Aguero-Rosenfeld ◽  
Abdallah Flaifel ◽  
John Colavito ◽  
Kenneth Inglima ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Viral Entry ◽  
Cell Types ◽  
Host Cells ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Neurologic Sequelae ◽  
Show Evidence ◽  
Novel Coronavirus ◽  
Transmembrane Serine Protease

Neurologic manifestations of the novel coronavirus SARS-CoV-2 infection have received wide attention, but the mechanisms remain uncertain. Here, we describe computational data from public domain RNA-seq datasets and cerebrospinal fluid data from adult patients with severe COVID-19 pneumonia that suggest that SARS-CoV-2 infection of the central nervous system is unlikely. We found that the mRNAs encoding the ACE2 receptor and the TMPRSS2 transmembrane serine protease, both of which are required for viral entry into host cells, are minimally expressed in the major cell types of the brain. In addition, CSF samples from 13 adult encephalopathic COVID-19 patients diagnosed with the viral infection via nasopharyngeal swab RT-PCR did not show evidence for the virus. This particular finding is robust for two reasons. First, the RT-PCR diagnostic was validated for CSF studies using stringent criteria; and second, 61% of these patients had CSF testing within 1 week of a positive nasopharyngeal diagnostic test. We propose that neurologic sequelae of COVID-19 are not due to SARS-CoV-2 meningoencephalitis and that other etiologies are more likely mechanisms.

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