scholarly journals A Quantitative and Kinetic Fusion Protein-Triggering Assay Can Discern Distinct Steps in the Nipah Virus Membrane Fusion Cascade

2010 ◽  
Vol 84 (16) ◽  
pp. 8033-8041 ◽  
Author(s):  
Hector C. Aguilar ◽  
Vanessa Aspericueta ◽  
Lindsey R. Robinson ◽  
Karen E. Aanensen ◽  
Benhur Lee
Keyword(s):  
Membrane Fusion ◽  
Receptor Binding ◽  
Nipah Virus ◽  
Heptad Repeat ◽  
Critical Parameters ◽  
Second Phase ◽  
Two Phase ◽  
Glycoprotein G ◽  
Fusion Glycoprotein ◽  
Functional Features

ABSTRACT The deadly paramyxovirus Nipah virus (NiV) contains a fusion glycoprotein (F) with canonical structural and functional features common to its class. Receptor binding to the NiV attachment glycoprotein (G) triggers F to undergo a two-phase conformational cascade: the first phase progresses from a metastable prefusion state to a prehairpin intermediate (PHI), while the second phase is marked by transition from the PHI to the six-helix-bundle hairpin. The PHI can be captured with peptides that mimic F's heptad repeat regions, and here we utilized a NiV heptad repeat peptide to quantify PHI formation and the half-lives (t 1/2) of the first and second fusion cascade phases. We found that ephrinB2 receptor binding to G triggered ∼2-fold more F than that triggered by ephrinB3, consistent with the increased rate and extent of fusion observed with ephrinB2- versus ephrinB3-expressing cells. In addition, for a series of hyper- and hypofusogenic F mutants, we quantified F-triggering capacities and measured the kinetics of their fusion cascade phases. Hyper- and hypofusogenicity can each be manifested through distinct stages of the fusion cascade, giving rise to vastly different half-lives for the first (t 1/2, 1.9 to 7.5 min) or second (t 1/2, 1.5 to 15.6 min) phase. While three mutants had a shorter first phase and a longer second phase than the wild-type protein, one mutant had the opposite phenotype. Thus, our results reveal multiple critical parameters that govern the paramyxovirus fusion cascade, and our assays should help efforts to elucidate other class I membrane fusion processes.

scholarly journals Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates both Early and Late Steps in the Membrane Fusion Cascade

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
J. Lizbeth Reyes Zamora ◽  
Victoria Ortega ◽  
Gunner P. Johnston ◽  
Jenny Li ◽  
Nicole M. André ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Host Range ◽  
Viral Entry ◽  
Nipah Virus ◽  
Fusion Process ◽  
Fusion Pore ◽  
Animal Pathogens ◽  
Fusion Glycoprotein ◽  
Helical Region

ABSTRACT Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members. IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens, such as measles, mumps, and parainfluenza viruses and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the central nervous system (CNS) and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40 to 100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. In this study, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.

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 Proteome Based Approach Defines Candidates for Designing a Multitope Vaccine against the Nipah Virus

2021 ◽  
Vol 22 (17) ◽  
pp. 9330 ◽  
Author(s):  
Mohamed A. Soltan ◽  
Muhammad Alaa Eldeen ◽  
Nada Elbassiouny ◽  
Ibrahim Mohamed ◽  
Dalia A. El-damasy ◽  
...  
Keyword(s):  
Nipah Virus ◽  
Epitope Prediction ◽  
Dynamics Simulation ◽  
World Health ◽  
Effective Vaccine ◽  
Emerging Viruses ◽  
Glycoprotein G ◽  
Fusion Glycoprotein ◽  
Physicochemical Features ◽  
Health Organization

Nipah virus is one of the most harmful emerging viruses with deadly effects on both humans and animals. Because of the severe outbreaks, in 2018, the World Health Organization focused on the urgent need for the development of effective solutions against the virus. However, up to date, there is no effective vaccine against the Nipah virus in the market. In the current study, the complete proteome of the Nipah virus (nine proteins) was analyzed for the antigenicity score and the virulence role of each protein, where we came up with fusion glycoprotein (F), glycoprotein (G), protein (V), and protein (W) as the candidates for epitope prediction. Following that, the multitope vaccine was designed based on top-ranking CTL, HTL, and BCL epitopes from the selected proteins. We used suitable linkers, adjuvant, and PADRE peptides to finalize the constructed vaccine, which was analyzed for its physicochemical features, antigenicity, toxicity, allergenicity, and solubility. The designed vaccine passed these assessments through computational analysis and, as a final step, we ran a docking analysis between the designed vaccine and TLR-3 and validated the docked complex through molecular dynamics simulation, which estimated a strong binding and supported the nomination of the designed vaccine as a putative solution for Nipah virus. Here, we describe the computational approach for design and analysis of this vaccine.

scholarly journals Two-Step Conformational Changes in a Coronavirus Envelope Glycoprotein Mediated by Receptor Binding and Proteolysis

2009 ◽  
Vol 83 (21) ◽  
pp. 11133-11141 ◽  
Author(s):  
Shutoku Matsuyama ◽  
Fumihiro Taguchi
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Hydrophobic Interactions ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Heptad Repeat ◽  
Soluble Form ◽  
S Protein

ABSTRACT The coronaviruses mouse hepatitis virus type 2 (MHV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV) utilize proteases to enter host cells. Upon receptor binding, the spike (S) proteins of both viruses are activated for membrane fusion by proteases, such as trypsin, present in the environment, facilitating virus entry from the cell surface. In contrast, in the absence of extracellular proteases, these viruses can enter cells via an endosomal pathway and utilize endosomal cathepsins for S protein activation. We demonstrate that the MHV-2 S protein uses multistep conformational changes for membrane fusion. After interaction with a soluble form of the MHV receptor (CEACAM1a), the metastable form of S protein is converted to a stable trimer, as revealed by mildly denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liposome-binding assays indicate that the receptor-bound virions are associated with the target membrane through hydrophobic interactions. The exposure of receptor-bound S protein to trypsin or cathepsin L (CPL) induces the formation of six-helix bundles (6HB), the final conformation. This trypsin- or CPL-mediated conversion to 6HB can be blocked by a heptad repeat peptide known to block the formation of 6HB. Although trypsin treatment enabled receptor-bound MHV-2 to enter from the cell surface, CPL failed to do so. Interestingly, consecutive treatment with CPL and then chlorpromazine enabled a portion of the virus to enter from cell surface. These results suggest that trypsin suffices for the induction of membrane fusion of receptor-primed S protein, but an additional unidentified cellular factor is required to trigger membrane fusion by CPL.

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

2008 ◽  
Vol 284 (3) ◽  
pp. 1628-1635 ◽  
Author(s):  
Hector C. Aguilar ◽  
Zeynep Akyol Ataman ◽  
Vanessa Aspericueta ◽  
Angela Q. Fang ◽  
Matthew Stroud ◽  
...  
Keyword(s):  
Nipah Virus ◽  
Virus Attachment ◽  
Glycoprotein G ◽  
Fusion Glycoprotein ◽  
Activation Site

A Comparison of Tem and Apfim to the Interpretation of Modulated Microstructures

1985 ◽  
Vol 43 ◽  
pp. 70-71
Author(s):  
M.G. Burke ◽  
M.K. Miller
Keyword(s):  
Low Temperature ◽  
Microstructural Characterization ◽  
Superlattice Reflection ◽  
High Volume ◽  
Atom Probe ◽  
Dark Field ◽  
Miscibility Gap ◽  
Second Phase ◽  
Two Phase ◽  
Probe Field

Interpretation of fine-scale microstructures containing high volume fractions of second phase is complex. In particular, microstructures developed through decomposition within low temperature miscibility gaps may be extremely fine. This paper compares the morphological interpretations of such complex microstructures by the high-resolution techniques of TEM and atom probe field-ion microscopy (APFIM).The Fe-25 at% Be alloy selected for this study was aged within the low temperature miscibility gap to form a <100> aligned two-phase microstructure. This triaxially modulated microstructure is composed of an Fe-rich ferrite phase and a B2-ordered Be-enriched phase. The microstructural characterization through conventional bright-field TEM is inadequate because of the many contributions to image contrast. The ordering reaction which accompanies spinodal decomposition in this alloy permits simplification of the image by the use of the centered dark field technique to image just one phase. A CDF image formed with a B2 superlattice reflection is shown in fig. 1. In this CDF micrograph, the the B2-ordered Be-enriched phase appears as bright regions in the darkly-imaging ferrite. By examining the specimen in a [001] orientation, the <100> nature of the modulations is evident.

THE DYNAMIC FRACTURE OF TWO PHASE ALLOYS WITH SECOND PHASE INCLUSIONS

1985 ◽  
Vol 46 (C5) ◽  
pp. C5-251-C5-255
Author(s):  
S. Pytel ◽  
L. Wojnar
Keyword(s):  
Dynamic Fracture ◽  
Second Phase ◽  
Two Phase
Sign in / Sign up

Export Citation Format

Share Document