scholarly journals Single virus assay reveals membrane determinants and mechanistic features of Sendai virus binding

2021 ◽  
Author(s):  
Amy Lam ◽  
Orville O Kirkland ◽  
Papa Freduah Anderson ◽  
Nandini Seetharaman ◽  
Dragan Vujovic ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Lipid Bilayers ◽  
Receptor Binding ◽  
Influenza A ◽  
Viral Vector ◽  
Sendai Virus ◽  
Structural Features ◽  
Host Cells ◽  
Cholesterol Concentration ◽  
Viral Fusion

Sendai virus (SeV, formally murine respirovirus) is a membrane-enveloped, negative sense RNA virus in the Paramyxoviridae family, and is closely related to human parainfluenza viruses. SeV has long been utilized as a model paramyxovirus and has recently gained attention as a viral vector candidate for both laboratory and clinical applications. To infect host cells, SeV must first bind to sialic-acid glycolipid or glycoprotein receptors on the host cell surface via its hemagglutinin-neuraminidase (HN) protein. Receptor binding induces a conformational change in HN, which allosterically triggers the viral fusion (F) protein to catalyze membrane fusion. While it is known that SeV binds to α2,3-linked sialic acid receptors, and there has been some study into the chemical requirements of those receptors, key mechanistic features of SeV binding remain unknown, in part because traditional approaches often convolve binding and fusion. Here, we develop and employ a fluorescence microscopy-based assay to observe SeV binding to supported lipid bilayers (SLBs) at the single particle level, which easily disentangles binding from fusion. Using this assay, we investigate mechanistic questions of SeV binding. We identify chemical structural features of ganglioside receptors that influence viral binding and demonstrate that binding is cooperative with respect to receptor density. We measure the characteristic decay time of unbinding and provide evidence supporting a "rolling" mechanism of viral mobility following receptor binding. We also study the dependence of binding on target cholesterol concentration. Interestingly, we find that while SeV binding shows striking parallels in cooperative binding with a prior report of Influenza A virus, it does not demonstrate a similar sensitivity to cholesterol concentration and receptor nano-cluster formation.

scholarly journals Potent sialic acid inhibitors that target influenza A virus hemagglutinin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yu-Jen Chang ◽  
Cheng-Yun Yeh ◽  
Ju-Chien Cheng ◽  
Yu-Qi Huang ◽  
Kai-Cheng Hsu ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Antiviral Activity ◽  
Binding Site ◽  
Influenza A Virus ◽  
Receptor Binding ◽  
Influenza A ◽  
The United States ◽  
Ligand Complex ◽  
Receptor Binding Site ◽  
Computational Strategy

AbstractEradicating influenza A virus (IAV) is difficult, due to its genetic drift and reassortment ability. As the infectious cycle is initiated by the influenza glycoprotein, hemagglutinin (HA), which mediates the binding of virions to terminal sialic acids moieties, HA is a tempting target of anti-influenza inhibitors. However, the complexity of the HA structure has prevented delineation of the structural characterization of the HA protein–ligand complex. Our computational strategy efficiently analyzed > 200,000 records of compounds held in the United States National Cancer Institute (NCI) database and identified potential HA inhibitors, by modeling the sialic acid (SA) receptor binding site (RBS) for the HA structure. Our modeling revealed that compound NSC85561 showed significant antiviral activity against the IAV H1N1 strain with EC50 values ranging from 2.31 to 2.53 µM and negligible cytotoxicity (CC50 > 700 µM). Using the NSC85561 compound as the template to generate 12 derivatives, robust bioassay results revealed the strongest antiviral efficacies with NSC47715 and NSC7223. Virtual screening clearly identified three SA receptor binding site inhibitors that were successfully validated in experimental data. Thus, our computational strategy has identified SA receptor binding site inhibitors against HA that show IAV-associated antiviral activity.

scholarly journals Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

2015 ◽  
Vol 89 (8) ◽  
pp. 4504-4516 ◽  
Author(s):  
Lauren Byrd-Leotis ◽  
Summer E. Galloway ◽  
Evangeline Agbogu ◽  
David A. Steinhauer
Keyword(s):  
Membrane Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Influenza A ◽  
Mutational Analysis ◽  
Host Cells ◽  
Influenza A Viruses ◽  
Pathogenic Potential ◽  
Additional Tool ◽  
Improved Stability

ABSTRACTInfluenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles.IMPORTANCEThe influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components.

scholarly journals Macrophage Receptors for Influenza A Virus: Role of the Macrophage Galactose-Type Lectin and Mannose Receptor in Viral Entry

2010 ◽  
Vol 84 (8) ◽  
pp. 3730-3737 ◽  
Author(s):  
Jacqueline P. Upham ◽  
Danielle Pickett ◽  
Tatsuro Irimura ◽  
E. Margot Anders ◽  
Patrick C. Reading
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Viral Entry ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Mannose Receptor ◽  
Host Cells ◽  
Cell Type ◽  
Carbohydrate Recognition Domains ◽  
Macrophage Populations

ABSTRACT Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca2+-dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.

scholarly journals A small-molecule fragment that emulates binding of receptor and broadly neutralizing antibodies to influenza A hemagglutinin

2018 ◽  
Vol 115 (16) ◽  
pp. 4240-4245 ◽  
Author(s):  
Rameshwar U. Kadam ◽  
Ian A. Wilson
Keyword(s):  
Influenza Virus ◽  
Small Molecule ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Binding Mode ◽  
Host Cells ◽  
Viral Pathogen ◽  
Group 2 ◽  
Group 1

The influenza virus hemagglutinin (HA) glycoprotein mediates receptor binding and membrane fusion during viral entry in host cells. Blocking these key steps in viral infection has applications for development of novel antiinfluenza therapeutics as well as vaccines. However, the lack of structural information on how small molecules can gain a foothold in the small, shallow receptor-binding site (RBS) has hindered drug design against this important target on the viral pathogen. Here, we report on the serendipitous crystallization-based discovery of a small-molecule N-cyclohexyltaurine, commonly known as the buffering agent CHES, that is able to bind to both group-1 and group-2 HAs of influenza A viruses. X-ray structural characterization of group-1 H5N1 A/Vietnam/1203/2004 (H5/Viet) and group-2 H3N2 A/Hong Kong/1/1968 (H3/HK68) HAs at 2.0-Å and 2.57-Å resolution, respectively, revealed that N-cyclohexyltaurine binds to the heart of the conserved HA RBS. N-cyclohexyltaurine mimics the binding mode of the natural receptor sialic acid and RBS-targeting bnAbs through formation of similar hydrogen bonds and CH-π interactions with the HA. In H3/HK68, N-cyclohexyltaurine also binds to a conserved pocket in the stem region, thereby exhibiting a dual-binding mode in group-2 HAs. These long-awaited structural insights into RBS recognition by a noncarbohydrate-based small molecule enhance our knowledge of how to target this important functional site and can serve as a template to guide the development of novel broad-spectrum small-molecule therapeutics against influenza virus.

scholarly journals Role of Neuraminidase in Influenza A(H7N9) Virus Receptor Binding

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Donald J. Benton ◽  
Stephen A. Wharton ◽  
Stephen R. Martin ◽  
John W. McCauley
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Binding Site ◽  
Influenza A Virus ◽  
Receptor Binding ◽  
Influenza A ◽  
Health Concern ◽  
H7n9 Virus ◽  
Virus Receptor ◽  
Sialic Acid Receptors

ABSTRACT Influenza A(H7N9) viruses have caused a large number of zoonotic infections since their emergence in 2013. They remain a public health concern due to the repeated high levels of infection with these viruses and their perceived pandemic potential. A major factor that determines influenza A virus fitness and therefore transmissibility is the interaction of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) with the cell surface receptor sialic acid. Typically, the HA is responsible for binding to the sialic acid to allow virus internalization and the NA is a sialidase responsible for cleaving sialic acid to aid virus spread and release. N9 NA has previously been shown to have receptor binding properties mediated by a sialic acid binding site, termed the hemadsorption (Hb) site, which is discrete from the enzymatically active sialidase site. This study investigated the N9 NA from a zoonotic H7N9 virus strain in order to determine its possible role in virus receptor binding. We demonstrate that this N9 NA has an active Hb site which binds to sialic acid, which enhances overall virus binding to sialic acid receptor analogues. We also show that the N9 NA can also contribute to receptor binding due to unusual kinetic characteristics of the sialidase site which specifically enhance binding to human-like α2,6-linked sialic acid receptors. IMPORTANCE The interaction of influenza A virus glycoproteins with cell surface receptors is a major determinant of infectivity and therefore transmissibility. Understanding these interactions is important for understanding which factors are necessary to determine pandemic potential. Influenza A viruses generally mediate binding to cell surface sialic acid receptors via the hemagglutinin (HA) glycoprotein, with the neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release. Previous studies showed that the NA proteins of the N9 subtype can bind sialic acid via a separate binding site distinct from the sialidase active site. This study demonstrates for purified protein and virus that the NA of the zoonotic H7N9 viruses has a binding capacity via both the secondary binding site and unusual kinetic properties of the sialidase site which promote receptor binding via this site and which enhance binding to human-like receptors. This could have implications for understanding human-to-human transmission of these viruses.

scholarly journals Molecular Aspects of Spike–ACE2 Interaction

Encyclopedia ◽  
2022 ◽  
Vol 2 (1) ◽  
pp. 96-108
Author(s):  
Luigi De Masi ◽  
Maria Antonia Argenio ◽  
Deborah Giordano ◽  
Angelo Facchiano
Keyword(s):  
Receptor Binding ◽  
Computational Analysis ◽  
Viral Vector ◽  
Cell Receptor ◽  
Viral Envelope ◽  
Host Cells ◽  
Angiotensin Converting Enzyme 2 ◽  
Binding Interface ◽  
Molecular Aspects ◽  
Distinguishing Features

A new betacoronavirus (CoV-2) is responsible for the pandemic of severe acute respiratory syndrome (SARS) that began in China at the end of 2019, today known as COronaVIrus Disease 2019 (COVID-19). Subsequent studies confirmed the human angiotensin-converting enzyme 2 (hACE2) as the main cell receptor of spike trimeric glycoprotein, located on the viral envelope, mediating the CoV-2 invasion into the host cells through the receptor-binding domain (RBD) of the spike. Computational analysis of the known experimental 3D structures of spike–ACE2 complexes evidenced distinguishing features in the molecular interactions at the RBD-cell receptor binding interface between CoV-2 and previous CoV-1. The spike represents a key target for drug design as well as an optimal antigen for RNA/viral vector vaccines and monoclonal antibodies in order to maximize prevention and therapy of COVID-19.

scholarly journals Single-Virus Investigation of Non-Sialic-Acid-Mediated Sendai Virus Binding to Supported Lipid Bilayers in the Absence of Ganglioside Receptor

2021 ◽  
Vol 120 (3) ◽  
pp. 320a
Author(s):  
Amy Lam ◽  
Nandini Seetharaman ◽  
Eunice Kim ◽  
Orville O. Kirkland ◽  
Robert J. Rawle
Keyword(s):  
Sialic Acid ◽  
Lipid Bilayers ◽  
Sendai Virus ◽  
Supported Lipid Bilayers ◽  
Virus Binding

scholarly journals The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus

2015 ◽  
Vol 90 (1) ◽  
pp. 206-221 ◽  
Author(s):  
Wy Ching Ng ◽  
Sarah L. Londrigan ◽  
Najla Nasr ◽  
Anthony L. Cunningham ◽  
Stuart Turville ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Influenza A Virus ◽  
Cho Cells ◽  
Influenza A ◽  
Chinese Hamster ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Infectious Entry ◽  
Attachment Factor

ABSTRACTIt is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5+) but not late (Rab7+) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor.IMPORTANCEOn the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and abone fideendocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin.

scholarly journals Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion

2010 ◽  
Vol 84 (9) ◽  
pp. 4366-4382 ◽  
Author(s):  
Nishi R. Sharma ◽  
Prashant Mani ◽  
Neha Nandwani ◽  
Rajakishore Mishra ◽  
Ajay Rana ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Viral Fusion ◽  
Reciprocal Regulation ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Syncytial Virus

ABSTRACT Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy.

scholarly journals Inhibiting Human Parainfluenza Virus Infection by Preactivating the Cell Entry Mechanism

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
S. F. Bottom-Tanzer ◽  
K. Rybkina ◽  
J. N. Bell ◽  
C. A. Alabi ◽  
C. Mathieu ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Conformational Changes ◽  
Host Cells ◽  
Parainfluenza Virus ◽  
Viral Fusion ◽  
Specific Antibodies ◽  
F Protein ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACTParamyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategyin vitro.Conformational changes in the F protein leading to adoption of the postfusion form of the protein—prior to receptor engagement of HN at the host cell membrane—render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevantex vivosystem, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCEParamyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategyin vitro. We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.

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