54 results on '"Aguilar, Hector C."'
Search Results
2. Antibody inhibition of measles virus entry.
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Aguilar, Hector C.
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MEASLES virus , *MEMBRANE fusion , *CELL receptors , *MEMBRANE glycoproteins , *VIRUS diseases - Abstract
The article focuses on the process of virus-cell membrane fusion, particularly in paramyxoviruses like measles virus (MeV), where two viral glycoproteins collaborate to enter host cells and induce syncytia formation. Zyla et al. used advanced cryo-electron microscopy to reveal a novel intermediate step in MeV fusion triggered by the antibody mAb 77, highlighting its potential as a therapeutic by inhibiting viral entry at a crucial stage.
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- 2024
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3. Flow virometry as a tool to study viruses.
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Zamora, J. Lizbeth Reyes and Aguilar, Hector C.
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FLOW cytometry , *DETECTION of microorganisms , *VIROLOGY , *VIRAL antigens , *VIRUS diseases , *GLYCOPROTEINS - Abstract
In the last few decades, flow cytometry has redefined the field of biology, exponentially enhancing our understanding of cells, immunology, and microbiology. Flow cytometry recently gave birth to flow virometry, a new way to detect, analyze, and characterize single viral particles. Detection of viruses by flow cytometry is possible due to improvements in current flow cytometers, calibration, and tuning methods. We summarize the recent birth and novel uses of flow virometry and the progressive evolution of this tool to advance the field of virology. We also discuss the various flow virometry methods used to identify and analyze viruses. We briefly summarize other applications of flow virometry, including: virus detection, quantification, population discrimination, and viral particles’ antigenic properties. Finally, we summarize how viral sorting will allow further progress of flow virometry to relate viral surface characteristics to infectivity properties. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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4. Finding proteases that make cells go viral.
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Aguilar, Hector C. and Buchholz, David W.
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INFLUENZA A virus , *HEMAGGLUTININ , *CELLS - Abstract
The activation of influenza virus hemagglutinin (HA) glycoprotein via cleavage by host cell proteases is essential for viral infectivity, and understanding the mechanisms for HA protein cleavage and how they may differ depending on the biological context is important for the development of flu treatments. However, the HA proteases involved in the activation of many viral strains remain unidentified. In this issue, Harbig et al. identify a repertoire of proteases that cleave HA and determine the proteases' functionality against specific HA glycoproteins. [ABSTRACT FROM AUTHOR]
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- 2020
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5. Triggering of the Newcastle Disease Virus Fusion Protein by a Chimeric Attachment Protein That Binds to Nipah Virus Receptors.
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Mirza, Anne M., Aguilar, Hector C., Qiyun Zhu, Mahon, Paul J., Rota, Paul A., Lee, Benhur, and Iorio, Ronald M.
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NEWCASTLE disease virus , *NIPAH virus , *PROTEINS , *HEMAGGLUTININ , *NEURAMINIDASE , *SIALIC acids , *CHIMERISM , *EPITOPES - Abstract
The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding. [ABSTRACT FROM AUTHOR]
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- 2011
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6. A Quantitative and Kinetic Fusion Protein-Triggering Assay Can Discern Distinct Steps in the Nipah Virus Membrane Fusion Cascade.
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Aguilar, Hector C., Aspericueta, Vanessa, Robinson, Lindsey R., Aanensen, Karen E., and Lee, Benhur
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PARAMYXOVIRUSES , *NIPAH virus , *GLYCOPROTEINS , *CELL membranes , *PHENOTYPES - 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 (t1/2) 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 (t1/2, 1.9 to 7.5 min) or second (t1/2, 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. [ABSTRACT FROM AUTHOR]
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- 2010
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7. Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation, Mobility, and Function to Block Syncytia Formation.
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Garner, Omai B., Aguilar, Hector C., Fulcher, Jennifer A., Levroney, Ernest L., Harrison, Rebecca, Wright, Lacey, Robinson, Lindsey R., Aspericueta, Vanessa, Panico, Maria, Haslam, Stuart M., Morris, Howard R., Dell, Anne, Lee, Benhur, and Baum, Linda G.
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NIPAH virus , *ENDOTHELIAL growth factors , *GLYCOPROTEINS , *VIRUS diseases , *CELL membranes , *PATHOLOGICAL physiology - Abstract
Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell. [ABSTRACT FROM AUTHOR]
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- 2010
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8. A Novel Receptor-induced Activation Site in the Nipah Virus Attachment Glycoprotein (G) Involved in Triggering the Fusion Glycoprotein (F).
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Aguilar, Hector C., Ataman, Zeynep Akyol, Aspericueta, Vanessa, Fang, Angela Q., Stroud, Matthew, Negrete, Oscar A., Kammerer, Richard A., and Lee, Benhur
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NIPAH virus , *PARAMYXOVIRUSES , *GLYCOPROTEINS , *RADIOLIGAND assay , *MONOCLONAL antibodies , *EPITOPES - Abstract
Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced all osteric activation site in Nipahvirus (NiV)G involved in triggering F-mediated fusion.Wefirst generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-GephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (β6S4/β1H1). Alanine scan mutants with in this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with Fat wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WTG, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding toWTG, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry. [ABSTRACT FROM AUTHOR]
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- 2009
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9. Cytoplasmic Tail of Moloney Murine Leukemia Virus Envelope Protein Influences the Conformation of the Extracellular Domain: Implications for Mechanisms of Action of the R Peptide.
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Aguilar, Hector C., Anderson, W. French, and Cannon, Paula M.
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MOUSE leukemia viruses , *PROTEIN precursors , *GLYCOPROTEINS - Abstract
The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptidetruncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein. [ABSTRACT FROM AUTHOR]
- Published
- 2003
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10. Adaptive immune cells are necessary for SARS-CoV-2-induced pathology.
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Imbiakha, Brian, Sahler, Julie M., Buchholz, David W., Ezzatpour, Shahrzad, Jager, Mason, Choi, Annette, Monreal, Isaac A., Byun, Haewon, Adeleke, Richard Ayomide, Leach, Justin, Whittaker, Gary, Dewhurst, Stephen, Rudd, Brian D., Aguilar, Hector C., and August, Avery
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B cells , *SARS-CoV-2 , *WEIGHT loss , *T cells - Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the ongoing global pandemic associated with morbidity and mortality in humans. Although disease severity correlates with immune dysregulation, the cellular mechanisms of inflammation and pathogenesis of COVID-19 remain relatively poorly understood. Here, we used mouse-adapted SARS-CoV-2 strain MA10 to investigate the role of adaptive immune cells in disease. We found that while infected wild-type mice lost ~10% weight by 3 to 4 days postinfection, rag-/- mice lacking B and T lymphocytes did not lose weight. Infected lungs at peak weight loss revealed lower pathology scores, fewer neutrophils, and lower interleukin-6 and tumor necrosis factor-a in rag-/- mice. Mice lacking aß T cells also had less severe weight loss, but adoptive transfer of T and B cells into rag-/- mice did not significantly change the response. Collectively, these findings suggest that while adaptive immune cells are important for clearing SARS-CoV-2 infection, this comes at the expense of increased inflammation and pathology. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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11. Characterization of the cellular lipid composition during SARS-CoV-2 infection.
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Abdel-Megied, Ahmed M., Monreal, Isaac A., Zhao, Limian, Apffel, Alex, Aguilar, Hector C., and Jones, Jace W.
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VIRAL envelope proteins , *TANDEM mass spectrometry , *SARS-CoV-2 , *LIPID metabolism , *LIPIDS , *VIRUS diseases - Abstract
Emerging and re-emerging zoonotic viral diseases continue to significantly impact public health. Of particular interest are enveloped viruses (e.g., SARS-CoV-2, the causative pathogen of COVID-19), which include emerging pathogens of highest concern. Enveloped viruses contain a viral envelope that encapsulates the genetic material and nucleocapsid, providing structural protection and functional bioactivity. The viral envelope is composed of a coordinated network of glycoproteins and lipids. The lipid composition of the envelope consists of lipids preferentially appropriated from host cell membranes. Subsequently, changes to the host cell lipid metabolism and an accounting of what lipids are changed during viral infection provide an opportunity to fingerprint the host cell's response to the infecting virus. To address this issue, we comprehensively characterized the lipid composition of VeroE6-TMPRSS2 cells infected with SARS-CoV-2. Our approach involved using an innovative solid-phase extraction technique to efficiently extract cellular lipids combined with liquid chromatography coupled to high-resolution tandem mass spectrometry. We identified lipid changes in cells exposed to SARS-CoV-2, of which the ceramide to sphingomyelin ratio was most prominent. The identification of a lipid profile (i.e., lipid fingerprint) that is characteristic of cellular SARS-CoV-2 infection lays the foundation for targeting lipid metabolism pathways to further understand how enveloped viruses infect cells, identifying opportunities to aid antiviral and vaccine development. [ABSTRACT FROM AUTHOR]
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- 2023
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12. Virus Entry by Endocytosis.
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Nicola, Anthony V., Aguilar, Hector C., Mercer, Jason, Ryckman, Brent, and Wiethoff, Christopher M.
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ENDOCYTOSIS , *VIRUS diseases , *VIRUS populations , *CELLULAR signal transduction , *VIRUSES , *CYTOPLASM , *SEROTYPES - Published
- 2013
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13. Interactions of Human Complement with Virus Particles Containing the Nipah Virus Glycoproteins.
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Johnson, John B., Aguilar, Hector C., Lee, Benhur, and Parks, Griffith D.
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NIPAH virus , *GLYCOPROTEINS , *IMMUNOGLOBULINS , *PARAMYXOVIRUSES , *ELECTRON microscopy , *MONOCLONAL antibodies - Abstract
Complement is an innate immune response system that most animal viruses encounter during natural infections. We have tested the role of human complement in the neutralization of virus particles harboring the Nipah virus (NiV) glycoproteins. A luciferase-expressing vesicular stomatitis virus (VSV) pseudotype that contained the NiV fusion (F) and attachment (G) glycoproteins (NiVpp) showed dose- and time-dependent activation of human complement through the alternative pathway. In contrast to our findings with other paramyxoviruses, normal human serum (NHS) alone did not neutralize NiVpp infectivity in vitro, and electron microscopy demonstrated no significant deposition of complement component C3 on particles. This lack of NiVpp neutralization by NHS was not due to a global inhibition of complement pathways, since complement was found to significantly enhance neutralization by antibodies specific for the NiV F and G glycoproteins. Complement components C4 and C1q were necessary but not sufficient by themselves for the enhancement of antibody neutralization. Human complement also enhanced NiVpp neutralization by a soluble version of the NiV receptor EphrinB2, and this depended on components in the classical pathway. The ability of complement to enhance neutralization fell into one of two profiles: (i) anti-F monoclonal antibodies showed enhancement only at high and not low antibody concentrations, and (ii) anti-G monoclonal antibodies and EphrinB2 showed enhancement at both high and very low levels of antibody (e.g., 3.1 ng) or EphrinB2 (e.g., 2.5 ng). Together, these data establish the importance of human complement in the neutralization of particles containing the NiV glycoproteins and will help guide the design of more effective therapeutics that harness the potency of complement pathways. [ABSTRACT FROM AUTHOR]
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- 2011
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14. Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling.
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Aguilar, Hector C., Matreyek, Kenneth A., Choi, Daniel Y., Filone, Claire Marie, Young, Sophia, and Benhur Lee
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PROTEINS , *VIRUSES , *NIPAH virus , *CELL fusion , *GENETIC mutation , *CYTOPLASMIC filaments - 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, r² = 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. [ABSTRACT FROM AUTHOR]
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- 2007
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15. N-Glycans on Nipah Virus Fusion Protein Protect against Neutralization but Reduce Membrane Fusion and Viral Entry.
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Aguilar, Hector C., Matreyek, Kenneth A., Filone, Claire Marie, Hashimi, Sara T., Levroney, Ernest L., Negrete, Oscar A., Bertolotti-Ciarlet, Andrea, Choi, Daniel Y., McHardy, Ian, Fulcher, Jennifer A., Su, Stephen V., Wolf, Mike C., Kohatsu, Luciana, Baum, Linda G., and Lee, Benhur
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PARAMYXOVIRUSES , *GLYCOPROTEINS , *IMMUNOGLOBULINS , *RNA viruses , *CELLS - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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16. Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely Virus, from Fruit Bats in Madagascar.
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Madera, Sharline, Kistler, Amy, Ranaivoson, Hafaliana C., Ahyong, Vida, Andrianiaina, Angelo, Andry, Santino, Raharinosy, Vololoniaina, Randriambolamanantsoa, Tsiry H., Ravelomanantsoa, NyAnjara Fifi, Tato, Cristina M., DeRisi, Joseph L., Aguilar, Hector C., Lacoste, Vincent, Dussart, Philippe, Heraud, Jean-Michel, and Brook, Cara E.
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EPHRIN receptors , *CYTOSKELETAL proteins , *BATS , *RNA editing , *FRUIT , *PARAMYXOVIRUSES - Abstract
The genus Henipavirus (family Paramyxoviridae) currently comprises seven viruses, four of which have demonstrated prior evidence of zoonotic capacity. These include the biosafety level 4 agents Hendra (HeV) and Nipah (NiV) viruses, which circulate naturally in pteropodid fruit bats. Here, we describe and characterize Angavokely virus (AngV), a divergent henipavirus identified in urine samples from wild, Madagascar fruit bats. We report the nearly complete 16,740-nucleotide genome of AngV, which encodes the six major henipavirus structural proteins (nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, and L polymerase). Within the phosphoprotein (P) gene, we identify an alternative start codon encoding the AngV C protein and a putative mRNA editing site where the insertion of one or two guanine residues encodes, respectively, additional V and W proteins. In other paramyxovirus systems, C, V, and W are accessory proteins involved in antagonism of host immune responses during infection. Phylogenetic analysis suggests that AngV is ancestral to all four previously described bat henipaviruses--HeV, NiV, Cedar virus (CedV), and Ghanaian bat virus (GhV)--but evolved more recently than rodent- and shrew-derived henipaviruses, Mojiang (MojV), Gamak (GAKV), and Daeryong (DARV) viruses. Predictive structure-based alignments suggest that AngV is unlikely to bind ephrin receptors, which mediate cell entry for all other known bat henipaviruses. Identification of the AngV receptor is needed to clarify the virus's potential host range. The presence of V and W proteins in the AngV genome suggest that the virus could be pathogenic following zoonotic spillover. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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17. An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity.
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Monticelli, Stephanie R., Bryk, Peter, Brewer, Matthew G., Aguilar, Hector C., Norbury, Christopher C., and Ward, Brian M.
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IMMUNE response , *VIRAL tropism , *MOLLUSCUM contagiosum , *VIRION , *VACCINIA , *PATHOGENESIS - Abstract
The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity. Author summary: Viral glycoproteins are critical determinants of host cell tropism, immunity, and pathogenesis. Vaccinia virus was used for the most successful immunization program in history, and poxviruses continue to be used as vaccine vectors. Here, we report that vaccinia virus extracellular virion (EV) protein F13 plays an important, previously unappreciated, role in controlling glycoprotein incorporation, and that there is a direct relationship between glycoprotein concentrations and subsequent infectivity. Crucially, manipulation of the EV glycoprotein concentrations altered pathogenesis and lethality in an in vivo infection model, but did not markedly alter the induced immune response. These results have important implications that inform the design of safer and more efficacious poxvirus-based vaccine vectors by altering glycoprotein content. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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18. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell–cell fusion
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Schowalter, Rachel M., Wurth, Mark A., Aguilar, Hector C., Lee, Benhur, Moncman, Carole L., McCann, Richard O., and Dutch, Rebecca Ellis
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PROTEINS , *BIOLOGICAL membranes , *GLYCOPROTEINS , *CELL membranes - Abstract
Abstract: The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell–cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell–cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1V12 or Cdc42V12 could increase cell–cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoAL63 decreased cell–cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia. [Copyright &y& Elsevier]
- Published
- 2006
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19. EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus.
- Author
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Negrete, Oscar A., Levroney, Ernest L., Aguilar, Hector C., Bertolotti-Ciarlet, Andrea, Nazarian, Ronen, Tajyar, Sara, and Lee, Benhur
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PARAMYXOVIRUSES , *PROTEINS , *NERVOUS system , *BRAIN diseases , *CLINICAL medicine , *AMINO acids , *PROTEIN-tyrosine kinases , *GENETIC transformation - Abstract
Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70 per cent of infected patients, and there is evidence of human–to–human transmission. Endothelial syncytia, comprised of multinucleated giant-endothelial cells, are frequently found in NiV infections, and are mediated by the fusion (F) and attachment (G) envelope glycoproteins. Identification of the receptor for this virus will shed light on the pathobiology of NiV infection, and spur the rational development of effective therapeutics. Here we report that ephrinB2, the membrane-bound ligand for the EphB class of receptor tyrosine kinases (RTKs), specifically binds to the attachment (G) glycoprotein of NiV. Soluble Fc-fusion proteins of ephrinB2, but not ephrinB1, effectively block NiV fusion and entry into permissive cell types. Moreover, transfection of ephrinB2 into non-permissive cells renders them permissive for NiV fusion and entry. EphrinB2 is expressed on endothelial cells and neurons, which is consistent with the known cellular tropism for NiV. Significantly, we find that NiV-envelope-mediated infection of microvascular endothelial cells and primary cortical rat neurons is inhibited by soluble ephrinB2, but not by the related ephrinB1 protein. Cumulatively, our data show that ephrinB2 is a functional receptor for NiV. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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20. Headless Henipaviral Receptor Binding Glycoproteins Reveal Fusion Modulation by the Head/Stalk Interface and Postreceptor Binding Contributions of the Head Domain.
- Author
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Yao Yu Yeo, Buchholz, David W., Gamble, Amandine, Jager, Mason, and Aguilar, Hector C.
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CELL fusion , *STALKING , *PHENOTYPES , *CARRIER proteins - Abstract
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 demonstrate 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 C-terminal stalk 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. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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- View/download PDF
21. 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.
- Author
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Zamora, J. Lizbeth Reyes, Ortega, Victoria, Johnston, Gunner P., Jenny Li, and Aguilar, Hector C.
- Subjects
- *
MEMBRANE fusion , *NIPAH virus , *CELL fusion , *PROTEIN conformation , *CHIMERIC proteins , *VIRAL genomes - Abstract
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 receptorbinding 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 heptad repeat 1 (HR1) and HR2 regions in the fusion protein prefusion conformation bind to form a six-helix bundle in the postfusion conformation. Here, structural comparisons between the F prefusion and postfusion 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 scanning 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 syncytium 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. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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22. Roles of Cholesterol in Early and Late Steps of the Nipah Virus Membrane Fusion Cascade.
- Author
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Contreras, Erik M., Johnston, Gunner P., Buchholz, David W., Ortega, Victoria, Monreal, I. Abrrey, Zamora, J. Lizbeth R., Cheung, Tracy, and Aguilar, Hector C.
- Subjects
- *
MEMBRANE fusion , *NIPAH virus , *CHOLESTEROL , *PARAMYXOVIRUSES , *CELL fusion , *VESICULAR stomatitis , *MUMPS - Abstract
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. Although cholesterol has been associated with the 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. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium 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 (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. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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23. Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates both Early and Late Steps in the Membrane Fusion Cascade.
- Author
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Zamora, J. Lizbeth Reyes, Ortega, Victoria, Johnston, Gunner P., Jenny Li, André, Nicole M., Monreal, I. Abrrey, Contreras, Erik M., Whittaker, Gary R., and Aguilar, Hector C.
- Subjects
- *
MEMBRANE fusion , *NIPAH virus , *CELL receptors , *CELL fusion , *PROTEIN conformation , *VIRAL envelopes , *CHIMERIC proteins - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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24. Feline Morbillivirus, a New Paramyxovirus Possibly Associated with Feline Kidney Disease.
- Author
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Choi, Eun Jin, Ortega, Victoria, and Aguilar, Hector C.
- Subjects
- *
KIDNEY diseases , *FELIDAE , *CHRONIC kidney failure , *MORBILLIVIRUSES , *CATS , *VIRAL tropism - Abstract
Feline morbillivirus (FeMV) was first isolated in stray cats in Hong Kong in 2012. Since its discovery, the virus has been reported in domestic cats worldwide, including in Hong Kong, Japan, Italy, US, Brazil, Turkey, UK, Germany, and Malaysia. FeMV is classified in the Morbillivirus genus within the Paramyxoviridae family. FeMV research has focused primarily on determining the host range, symptoms, and characteristics of persistent infections in vitro. Importantly, there is a potential association between FeMV infection and feline kidney diseases, such as tubulointerstitial nephritis (TIN) and chronic kidney diseases (CKD), which are known to significantly affect feline health and survival. However, the tropism and viral entry mechanism(s) of FeMV remain unknown. In this review, we summarize the FeMV studies up to date, including the discoveries of various FeMV strains, basic virology, pathogenicity, and disease signs. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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25. Modified Sialic Acids on Mucus and Erythrocytes Inhibit Influenza A Virus Hemagglutinin and Neuraminidase Functions.
- Author
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Barnard, Karen N., Alford-Lawrence, Brynn K., Buchholz, David W., Wasik, Brian R., LaClair, Justin R., Hai Yu, Honce, Rebekah, Ruhl, Stefan, Pajic, Petar, Daugherity, Erin K., Xi Chen, Schultz-Cherry, Stacey L., Aguilar, Hector C., Varki, Ajit, and Parrish, Colin R.
- Subjects
- *
NEURAMINIDASE , *SIALIC acids , *INFLUENZA A virus , *MUCUS , *ERYTHROCYTES , *HEMAGGLUTININ - Abstract
Sialic acids (Sia) are the primary receptors for influenza viruses and are widely displayed on cell surfaces and in secreted mucus. Sia may be present in variant forms that include O-acetyl modifications at C-4, C-7, C-8, and C-9 positions and N-acetyl or N-glycolyl at C-5. They can also vary in their linkages, including -2-3 or -2-6 linkages. Here, we analyze the distribution of modified Sia in cells and tissues of wild-type mice or in mice lacking CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme, which synthesizes N-glycolyl (Neu5Gc) modifications. We also examined the variation of Sia forms on erythrocytes and in saliva from different animals. To determine the effect of Sia modifications on influenza A virus (IAV) infection, we tested for effects on hemagglutinin (HA) binding and neuraminidase (NA) cleavage. We confirmed that 9-O-acetyl, 7,9-O-acetyl, 4-O-acetyl, and Neu5Gc modifications are widely but variably expressed in mouse tissues, with the highest levels detected in the respiratory and gastrointestinal (GI) tracts. Secreted mucins in saliva and surface proteins of erythrocytes showed a high degree of variability in display of modified Sia between different species. IAV HAs from different virus strains showed consistently reduced binding to both Neu5Gc- and O-acetyl-modified Sia; however, while IAV NAs were inhibited by Neu5Gc and O-acetyl modifications, there was significant variability between NA types. The modifications of Sia in mucus may therefore have potent effects on the functions of IAV and may affect both pathogens and the normal flora of different mucosal sites. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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- View/download PDF
26. Nipah and Hendra viral glycoproteins induce comparable homologous but distinct heterologous fusion phenotypes.
- Author
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Bradel-Tretheway, Birgit G., Zamora, J. Lizbeth Reyes, Stone, Jacquelyn A., Liu, Qian, Jenny Li, and Aguilar, Hector C.
- Subjects
- *
VIRAL envelope proteins , *MEMBRANE glycoproteins , *P-glycoprotein , *GLYCOPROTEINS , *CELL fusion , *PHENOTYPES , *MEMBRANE fusion , *NIPAH virus - Abstract
Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are BSL-4 paramyxoviruses in the growing genus, Henipavirus. The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, pathognomonic of henipaviral infections. Here we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (HA tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F/G interactions, supporting the F/G dissociation model of henipaviral-induced membrane fusion, even in the context of heterologous glycoprotein pairs. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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- View/download PDF
27. Addicted to sugar: roles of glycans in the order Mononegavirales.
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Ortega, Victoria, Stone, Jacquelyn A, Contreras, Erik M, Iorio, Ronald M, and Aguilar, Hector C
- Subjects
- *
PROTEIN analysis , *GLYCANS , *MONONEGAVIRALES , *SUGAR analysis , *GLYCOSYLATION , *AMINO acid residues , *HOSTS (Biology) , *CARBOHYDRATE analysis - Abstract
Glycosylation is a biologically important protein modification process by which a carbohydrate chain is enzymatically added to a protein at a specific amino acid residue. This process plays roles in many cellular functions, including intracellular trafficking, cell–cell signaling, protein folding and receptor binding. While glycosylation is a common host cell process, it is utilized by many pathogens as well. Protein glycosylation is widely employed by viruses for both host invasion and evasion of host immune responses. Thus better understanding of viral glycosylation functions has potential applications for improved antiviral therapeutic and vaccine development. Here, we summarize our current knowledge on the broad biological functions of glycans for the Mononegavirales, an order of enveloped negative-sense single-stranded RNA viruses of high medical importance that includes Ebola, rabies, measles and Nipah viruses. We discuss glycobiological findings by genera in alphabetical order within each of eight Mononegavirales families, namely, the bornaviruses, filoviruses, mymonaviruses, nyamiviruses, paramyxoviruses, pneumoviruses, rhabdoviruses and sunviruses. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
28. Broad-spectrum antiviral JL122 blocks infection and inhibits transmission of aquatic rhabdoviruses.
- Author
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Balmer, Bethany F., Getchell, Rodman G., Powers, Rachel L., Lee, Jihye, Zhang, Tinghu, Jung, Michael E., Purcell, Maureen K., Snekvik, Kevin, and Aguilar, Hector C.
- Subjects
- *
RHABDOVIRUSES , *VIRAL transmission , *AQUATIC microbiology , *ANTIVIRAL agents , *VIRAL envelopes - Abstract
Abstract The aquaculture industry is growing rapidly to meet the needs for global protein consumption. Viral diseases in aquaculture are quite challenging due to lack of treatment options as well as limited injection-delivery vaccines, which are costly. Thus, water-immersion antiviral treatments are highly desirable. This study focused on broad-spectrum, light-activated antivirals that target the viral membrane (envelope) of viruses to prevent viral-cell membrane fusion, ultimately blocking viral entry into cells. Of the tested small-molecules, JL122, a new broad-spectrum antiviral previously unexplored against aquatic viruses, blocked infection of three aquatic rhabdoviruses (IHNV, VHSV and SVCV) in cell culture and in two live fish challenge models. Importantly, JL122 inhibited transmission of IHNV from infected to uninfected rainbow trout. Further, the effective antiviral concentrations were not toxic to cells or susceptible fish. These results show promise for JL122 to become an immersion treatment option for outbreaks of aquatic enveloped viral infections. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
29. Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress.
- Author
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Johnston, Gunner P., Contreras, Erik M., Dabundo, Jeffrey, Henderson, Bryce A., Matz, Keesha M., Ortega, Victoria, Ramirez, Alfredo, Park, Arnold, and Aguilar, Hector C.
- Subjects
- *
NIPAH virus , *VIRUS-like particles , *GLYCOPROTEINS , *ENDOCYTOSIS , *VIRAL proteins - Abstract
Nipah virus (NiV), a paramyxovirus in the genus Henipavirus, has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F) and, to a much lesser extent, the attachment glycoprotein (G) autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXØ or dityrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
30. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.
- Author
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AlHajri, Salim M., Cunha, Cristina W., Nicola, Anthony V., Aguilar, Hector C., Hong Li, and Tausa, Naomi S.
- Subjects
- *
HERPESVIRUS diseases , *GLYCOPROTEINS , *CELL membranes , *SHEEP , *MALIGNANT catarrhal fever - Abstract
Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism. IMPORTANCE: Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular factors necessary for virus-cell membrane fusion. We found that OvHV-2 glycoproteins B, H, and L are sufficient for, and viral glycoprotein Ov8 can significantly enhance, cell-cell membrane fusion. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
31. Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry.
- Author
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Weed, Darin J., Pritchard, Suzanne M., Gonzalez, Floricel, Aguilar, Hector C., and Nicola, Anthony V.
- Subjects
- *
HERPES simplex virus , *GLYCOPROTEINS , *ENDOCYTOSIS , *CELL surface antigens , *HEPARAN sulfate , *MONOCLONAL antibodies - Abstract
Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions. IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
32. Inhibition of an Aquatic Rhabdovirus Demonstrates Promise of a Broad-Spectrum Antiviral for Use in Aquaculture.
- Author
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Balmer, Bethany F., Powers, Rachel L., Ting-Hu Zhang, Lee, Jihye, Vigant, Frederic, Lee, Benhur, Jung, Michael E., Purcell, Maureen K., Snekvik, Kevin, and Aguilar, Hector C.
- Subjects
- *
RHABDOVIRUSES , *ANTIVIRAL agents , *AQUACULTURE , *PHOSPHOLIPIDS , *BILAYER lipid membranes - Abstract
Many enveloped viruses cause devastating disease in aquaculture, resulting in significant economic impact. LJ001 is a broad-spectrum antiviral compound that inhibits enveloped virus infections by specifically targeting phospholipids in the lipid bilayer via the production of singlet oxygen (¹O2). This stabilizes positive curvature and decreases membrane fluidity, which inhibits virus-cell membrane fusion during viral entry. Based on data from previous mammalian studies and the requirement of light for the activation of LJ001, we hypothesized that LJ001 may be useful as a preventative and/or therapeutic agent for infections by enveloped viruses in aquaculture. Here, we report that LJ001 was more stable with a prolonged inhibitory half-life at relevant aquaculture temperatures (15°C), than in mammalian studies at 37°C. When LJ001 was preincubated with our model virus, infectious hematopoietic necrosis virus (IHNV), infectivity was significantly inhibited in vitro (using the epithelioma papulosum cyprini [EPC] fish cell line) and in vivo (using rainbow trout fry) in a dose-dependent and time-dependent manner. While horizontal transmission of IHNV in a static cohabitation challenge model was reduced by LJ001, transmission was not completely blocked at established antiviral doses. Therefore, LJ001 may be best suited as a therapeutic for aquaculture settings that include viral infections with lower virus-shedding rates than IHNV or where higher viral titers are required to initiate infection of naive fish. Importantly, our data also suggest that LJ001-inactivated IHNV elicited an innate immune response in the rainbow trout host, making LJ001 potentially useful for future vaccination approaches. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
33. Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins.
- Author
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Stone, Jacquelyn A., Vemulapati, Bhadra M., Bradel-Tretheway, Birgit, and Aguilar, Hector C.
- Subjects
- *
NIPAH virus , *GLYCOPROTEINS , *PARAINFLUENZA viruses , *CELL communication , *IMMUNOPRECIPITATION - Abstract
The 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 proteinprotein 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. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
34. Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family.
- Author
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Stone, Jacquelyn A., Nicola, Anthony V., Baum, Linda G., and Aguilar, Hector C.
- Subjects
- *
GLYCANS , *HENIPAVIRUSES , *GLYCOSYLATION , *GLYCOPROTEINS , *CELL fusion , *NIPAH virus - Abstract
O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
35. Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.
- Author
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Xu, Kai, Chan, Yee-Peng, Bradel-Tretheway, Birgit, Akyol-Ataman, Zeynep, Zhu, Yongqun, Dutta, Somnath, Yan, Lianying, Feng, YanRu, Wang, Lin-Fa, Skiniotis, Georgios, Lee, Benhur, Zhou, Z. Hong, Broder, Christopher C., Aguilar, Hector C., and Nikolov, Dimitar B.
- Subjects
- *
CRYSTAL structure research , *CRYSTALLOGRAPHY , *NIPAH virus , *PARAMYXOVIRUSES , *GLYCOPROTEINS - Abstract
Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
36. Polyethylene glycol-mediated fusion of herpes simplex type 1 virions with the plasma membrane of cells that support endocytic entry.
- Author
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Walker, Erik B., Pritchard, Suzanne M., Cunha, Cristina W., Aguilar, Hector C., and Nicola, Anthony V.
- Subjects
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HERPES simplex , *CELL membranes , *POLYETHYLENE glycol , *GENE expression in viruses , *PROTEOLYSIS , *DNA replication , *ACIDIFICATION , *VIRUSES - Abstract
Background: Mouse B78 cells and Chinese hamster ovary (CHO) cells are important to the study of HSV-1 entry because both are resistant to infection at the level of viral entry. When provided with a gD-receptor such as nectin-1, these cells support HSV-1 entry by an endocytosis pathway. Treating some viruses bound to cells with the fusogen polyethylene glycol (PEG) mediates viral fusion with the cell surface but is insufficient to rescue viral entry. It is unclear whether PEG-mediated fusion of HSV with the plasma membrane of B78 or CHO cells results in successful entry and infection. Findings: Treating HSV-1 bound to B78 or CHO cells with PEG allowed viral entry as measured by virus-induced beta-galactosidase activity. Based on the mechanism of PEG action, we propose that entry likely proceeds by direct fusion of HSV particles with the plasma membrane. Under the conditions tested, PEG-mediated infection of CHO cells progressed to the level of HSV late gene expression, while B78 cells supported HSV DNA replication. We tested whether proteolysis or acidification of cell-bound virions could trigger HSV fusion with the plasma membrane. Under the conditions tested, mildly acidic pH of 5-6 or the protease trypsin were not capable of triggering HSV-1 fusion as compared to PEG-treated cell-bound virions. Conclusions: B78 cells and CHO cells, which typically endocytose HSV prior to viral penetration, are capable of supporting HSV-1 entry via direct penetration. HSV capsids delivered directly to the cytosol at the periphery of these cells complete the entry process. B78 and CHO cells may be utilized to screen for factors that trigger entry as a consequence of fusion of virions with the cell surface, and PEG treatment can provide a necessary control. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
37. Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene.
- Author
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Cunha, Cristina W., Taylor, Kathryne E., Pritchard, Suzanne M., Delboy, Mark G., Komala Sari, Tri, Aguilar, Hector C., Mossman, Karen L., and Nicola, Anthony V.
- Subjects
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HERPES simplex virus , *VIRAL genetics , *DELETION mutation , *GLYCOPROTEINS , *PHOSPHOPROTEINS , *PROTEIN expression , *VIRUSES - Abstract
Herpes simplex virus 1 (HSV-1) ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
38. Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus.
- Author
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Bradel-Tretheway, Birgit G., Qian Liu, Stone, Jacquelyn A., McInally, Samantha, and Aguilar, Hector C.
- Subjects
- *
GLYCANS , *NIPAH virus , *EPHRIN receptors , *MEMBRANE fusion , *GLYCOSYLATED hemoglobin , *CELL communication , *MOLECULAR immune response - Abstract
Hendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. 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. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
39. Branched dimerization of Tat peptide improves permeability to HeLa and hippocampal neuronal cells.
- Author
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Monreal, I. Abrrey, Liu, Qian, Tyson, Katherine, Bland, Tyler, Dalisay, Doralyn S., Adams, Erin V., Wayman, Gary A., Aguilar, Hector C., and Saludes, Jonel P.
- Subjects
- *
NEURON analysis , *TAT protein , *CELLULAR signal transduction , *DIMERIZATION , *CONFOCAL microscopy , *PERMEABILITY - Abstract
A dimeric branched peptide TATp-D designed as an analogue of the HIV-Tat protein transduction domain (TATp), a prototypical cell penetrating peptide (CPP), demonstrates significantly enhanced cell uptake at 0.25 to 2.5 μM. Live cell confocal laser scanning microscopy revealed that multivalency dramatically improved the permeation potency of TATp-D to HeLa and primary hippocampal neuronal cells. The observed enhanced ability of TATp-D to translocate through the membrane is highlighted by a non-linear dependence on concentration, exhibiting the greatest uptake at sub-micromolar concentrations as compared to TATp. Multimerization via bis-Fmoc Lysine offered a synthetically straightforward method to investigate the effects of multivalent CPPs while offering orthogonal handles for cargo attachment, increasing the utility of CPPs at significantly lower concentrations. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
40. Timing of Galectin-1 Exposure Differentially Modulates Nipah Virus Entry and Syncytium Formation in Endothelial Cells.
- Author
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Garner, Omai B., Yun, Tatyana, Pernet, Olivier, Aguilar, Hector C., Park, Arnold, Bowden, Thomas A., Freiberg, Alexander N., Lee, Benhur, and Bauma, Linda G.
- Subjects
- *
GALECTINS , *NIPAH virus , *ENDOTHELIAL cells , *PARAMYXOVIRUSES , *VIRUS research - Abstract
Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
41. Antibodies to ovine herpesvirus 2 glycoproteins decrease virus infectivity and prevent malignant catarrhal fever in rabbits.
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Cunha, Cristina W., Knowles, Donald P., Taus, Naomi S., O’Toole, Donal, Nicola, Anthony V., Aguilar, Hector C., and Li, Hong
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- *
IMMUNOGLOBULINS , *GLYCOPROTEINS , *VIRUS diseases , *FEVER , *LABORATORY rabbits , *ETIOLOGY of diseases , *PREVENTION - Abstract
Ovine herpesvirus-2 (OvHV-2) is the etiological agent of sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease of many species in the order Artiodactyla. Development of a vaccine is critical to prevent mortality. Because OvHV-2 has not been cultured in vitro , SA-MCF research is hindered by the lack of in vitro tools to study viral constituents and specific host immune responses. As an alternative, in this study the neutralizing activity of antibodies against OvHV-2 glycoproteins gB and gH/gL was evaluated in vivo using rabbits. OvHV-2-specific antibodies were developed in rabbits by immunization using biolistic delivery of plasmids expressing the genes of interest. A lethal dose of OvHV-2 was incubated with the antisera and then nebulized into rabbits. Virus neutralization was assessed by measuring infection parameters associated with the virus infectious dose. Anti-gB or anti-gH/gL antibodies alone blocked infection in five out of six rabbits (83%), while a combination of anti-gB and anti-gH/gL antibodies protected all six rabbits (100%) from infection. These results indicate that antibodies to OvHV-2 gB and gH/gL are capable of neutralizing virions, and consequently, reduce virus infectivity and prevent SA-MCF in rabbits. Thus, OvHV-2 gB and gH/gL are suitable targets to be tested in a SA-MCF vaccine aimed at stimulating neutralizing antibody responses. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
42. Nipah Virus Attachment Glycoprotein Stalk C-Terminal Region Links Receptor Binding to Fusion Triggering.
- Author
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Qian Liu, Bradel-Tretheway, Birgit, Monreal, Abrrey I., Saludes, Jonel P., Xiaonan Lu, Nicola, Anthony V., and Aguilar, Hector C.
- Subjects
- *
MEMBRANE fusion , *PARAMYXOVIRUSES , *CELL fusion , *RESPIRATORY syncytial virus , *VIRUS diseases - Abstract
Membrane 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. The emergent deadly viruses Nipah virus (NiV) and Hendra virus belong to the Henipavirus genus in the Paramyxoviridae family. 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. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
43. Nipah Virion Entry Kinetics, Composition, and Conformational Changes Determined by Enzymatic Virus-Like Particles and New Flow Virometry Tools.
- Author
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Landowski, Matthew, Dabundo, Jeffrey, Qian Liu, Nicola, Anthony V., and Aguilar, Hector C.
- Subjects
- *
NIPAH virus , *CELL membranes , *VIRUS diseases , *CELL fusion , *PARAMYXOVIRUSES - Abstract
Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. Drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and virus-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a β-lactamase (βLa) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By preloading target cells with the βLa fluorescent substrate CCF2- AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
44. Molecular Requirement for Sterols in Herpes Simplex Virus Entry and Infectivity.
- Author
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Wudiri, George A., Pritchard, Suzanne M., Hong Li, Jin Liu, Aguilar, Hector C., Gilk, Stacey D., and Nicola, Anthony V.
- Subjects
- *
HERPES simplex virus , *CHOLESTEROL , *MOLECULAR biology , *VIRION , *CELL membranes , *HYDROCARBONS - Abstract
Herpes simplex virus 1 (HSV-1) required cholesterol or desmosterol for virion-induced membrane fusion. HSV successfully entered DHCR24-/- cells, which lack a desmosterol-to-cholesterol conversion enzyme, indicating that entry can occur independently of cholesterol. Depletion of desmosterol from these cells resulted in diminished HSV-1 entry, suggesting a general sterol requirement for HSV-1 entry and that desmosterol can operate in virus entry. Cholesterol functioned more effectively than desmosterol, suggesting that the hydrocarbon tail of cholesterol influences viral entry. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
45. Fluorescent Probes Based on Nucleophilic Substitution-Cyclization for Hydrogen Sulfide Detection and Bioimaging.
- Author
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Peng, Bo, Chen, Wei, Liu, Chunrong, Rosser, Ethan W., Pacheco, Armando, Zhao, Yu, Aguilar, Hector C., and Xian, Ming
- Subjects
- *
FLUORESCENT probes , *NUCLEOPHILIC substitution reactions , *RING formation (Chemistry) , *HYDROGEN sulfide , *BIO-imaging sensors - Abstract
The design, synthesis, properties, and cell imaging applications of a series of 2-pyridyl disulfide based fluorescent probes (WSP1, WSP2, WSP3, WSP4 and WSP5) for hydrogen sulfide detection are reported. The strategy is based on the dual-nucleophilicity of hydrogen sulfide. A hydrogen sulfide mediated tandem nucleophilic substitution-cyclization reaction is used to release the fluorophores and turn on the fluorescence. The probes showed high sensitivity and selectivity for hydrogen sulfide over other reactive sulfur species, including cysteine and glutathione. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
46. Contributions of Herpes Simplex Virus 1 Envelope Proteins to Entry by Endocytosis.
- Author
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Sari, Tri Komala, Pritchard, Suzanne M., Cunha, Cristina W., Wudiri, George A., Laws, Elizabeth I., Aguilar, Hector C., Taus, Naomi S., and Nicola, Anthony V.
- Subjects
- *
HERPES simplex virus , *VIRAL envelope proteins , *ENDOCYTOSIS , *GLYCOPROTEINS , *PH effect - Abstract
Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the required HSV glycoproteins, gB, gD, and gH-gL, may be sufficient for entry regardless of entry route taken. This may be distinct from entry mechanisms employed by other human herpesviruses. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
47. Unraveling a Three-Step Spatiotemporal Mechanism of Triggering of Receptor-Induced Nipah Virus Fusion and Cell Entry.
- Author
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Liu, Qian, Stone, Jacquelyn A., Bradel-Tretheway, Birgit, Dabundo, Jeffrey, Benavides Montano, Javier A., Santos-Montanez, Jennifer, Biering, Scott B., Nicola, Anthony V., Iorio, Ronald M., Lu, Xiaonan, and Aguilar, Hector C.
- Subjects
- *
SPATIOTEMPORAL processes , *MEMBRANE fusion , *CELL adhesion , *CELL communication , *CELL junctions - Abstract
Membrane fusion is essential for entry of the biomedically-important paramyxoviruses into their host cells (viral-cell fusion), and for syncytia formation (cell-cell fusion), often induced by paramyxoviral infections [e.g. those of the deadly Nipah virus (NiV)]. For most paramyxoviruses, membrane fusion requires two viral glycoproteins. Upon receptor binding, the attachment glycoprotein (HN/H/G) triggers the fusion glycoprotein (F) to undergo conformational changes that merge viral and/or cell membranes. However, a significant knowledge gap remains on how HN/H/G couples cell receptor binding to F-triggering. Via interdisciplinary approaches we report the first comprehensive mechanism of NiV membrane fusion triggering, involving three spatiotemporally sequential cell receptor-induced conformational steps in NiV-G: two in the head and one in the stalk. Interestingly, a headless NiV-G mutant was able to trigger NiV-F, and the two head conformational steps were required for the exposure of the stalk domain. Moreover, the headless NiV-G prematurely triggered NiV-F on virions, indicating that the NiV-G head prevents premature triggering of NiV-F on virions by concealing a F-triggering stalk domain until the correct time and place: receptor-binding. Based on these and recent paramyxovirus findings, we present a comprehensive and fundamentally conserved mechanistic model of paramyxovirus membrane fusion triggering and cell entry. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
48. A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion.
- Author
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Vigant, Frederic, Jihye Lee, Hollmann, Axel, Tanner, Lukas B., Ataman, Zeynep Akyol, Yun, Tatyana, Guanghou Shui, Aguilar, Hector C., Dong Zhang, Meriwether, David, Roman-Sosa, Gleyder, Robinson, Lindsey R., Juelich, Terry L., Buczkowski, Hubert, Sunwen Chou, Castanho, Miguel A. R. B., Wolf, Mike C., Smith, Jennifer K., Banyard, Ashley, and Kielian, Margaret
- Subjects
- *
ANTIVIRAL agents , *ANTI-infective agents , *CELL fusion , *CYTOGENETICS , *THIAZOLIDINEDIONES - Abstract
LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (¹O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated ¹O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. ¹O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced ¹O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50 < 10 µM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of ¹O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
49. Individual N-Glycans Added at Intervals along the Stalk of the Nipah Virus G Protein Prevent Fusion but Do Not Block the Interaction with the Homologous F Protein.
- Author
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Qiyun Zhu, Biering, Scott B., Mirza, Anne M., Grasseschi, Brittany A., Mahon, Paul J., Lee, Benhur, Aguilar, Hector C., and Iorio, Ronald M.
- Subjects
- *
PARAMYXOVIRUSES , *PROTEINS , *NEWCASTLE disease virus , *HEMAGGLUTININ , *NEURAMINIDASE , *MEASLES virus , *NIPAH virus , *GLYCOPROTEINS - Abstract
The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven introduced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the ability of the G protein to complement the homologous F protein in the promotion of fusion. The most membrane-proximal added N-glycan reduces fusion by 80%. However, unlike similar NDV HN and MV H mutants, the NiV G glycosylation stalk mutants retain the ability to bind F, indicating that the fusion deficiency of these mutants is not due to prevention of the G-F interaction. These findings suggest that the G-F interaction is not mediated entirely by the stalk domain of G and may be more complex than that of HN/H-F. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
50. Cysteines in the Stalk of the Nipah Virus G Glycoprotein Are Located in a Distinct Subdomain Critical for Fusion Activation.
- Author
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Maar, Dianna, Harmon, Brooke, Chu, David, Schulz, Belinda, Aguilar, Hector C., Lee, Benhur, and Negrete, Oscar A.
- Subjects
- *
CYSTEINE , *NIPAH virus , *GLYCOPROTEINS , *PARAMYXOVIRUSES , *MEMBRANE fusion , *DIMERIZATION , *EPITOPES - Abstract
Paramyxoviruses initiate entry through the concerted action of the tetrameric attachment glycoprotein (HN, H, or G) and the trimeric fusion glycoprotein (F). The ectodomains of HN/H/G contain a stalk region important for oligomeric stability and for the F triggering resulting in membrane fusion. Paramyxovirus HN, H, and G form a dimer-of-dimers consisting of disulfidelinked dimers through their stalk domain cysteines. The G attachment protein stalk domain of the highly pathogenic Nipah virus (NiV) contains a distinct but uncharacterized cluster of three cysteine residues (C146, C158, C162). On the basis of a panoply of assays, we report that C158 and C162 of NiV-G likely mediate covalent subunit dimerization, while C146 mediates the stability of higher-order oligomers. For HN or H, mutation of stalk cysteines attenuates but does not abrogate the ability to trigger fusion. In contrast, the NiV-G stalk cysteine mutants were completely deficient in triggering fusion, even though they could still bind the ephrinB2 receptor and associate with F. Interestingly, all cysteine stalk mutants exhibited constitutive exposure of the Mab45 receptor binding-enhanced epitope, previously implicated in F triggering. The enhanced binding of Mab45 to the cysteine mutants relative to wild-type NiV-G, without the addition of the receptor, implicates the stalk cysteines in the stabilization of a pre-receptor-bound conformation and the regulation of F triggering. Sequence alignments revealed that the stalk cysteines were adjacent to a proline-rich microdomain unique to the Henipavirus genus. Our data propose that the cysteine cluster in the NiV-G stalk functions to maintain oligomeric stability but is more importantly involved in stabilizing a unique microdomain critical for triggering fusion. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
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