The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies

ABSTRACT The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae , facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism. IMPORTANCE Filoviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The present study shows that LLOV, like EBOV, counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction.

Download Full-text

A Multifactorial Analysis of Ebola Virus Glycoprotein Receptor Binding Domain

Journal of Medical Microbiology & Diagnosis ◽

10.4172/2161-0703.1000217 ◽

2016 ◽

Vol 05 (01) ◽

Author(s):

Joel K Weltman

Keyword(s):

Receptor Binding ◽

Ebola Virus ◽

Binding Domain ◽

Receptor Binding Domain ◽

Multifactorial Analysis ◽

Virus Glycoprotein

Download Full-text

SARS-CoV-2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability

10.1101/2020.03.21.001933 ◽

2020 ◽

Cited By ~ 4

Author(s):

Vinicio Armijos-Jaramillo ◽

Justin Yeager ◽

Claire Muslin ◽

Yunierkis Perez-Castillo

Keyword(s):

Receptor Binding ◽

Evolutionary Dynamics ◽

Hot Spot ◽

Critical Role ◽

Human Cells ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Salt Bridges

AbstractThe emergence of SARS-CoV-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. This novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the SARS-CoV cell receptor hACE2. In the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. Next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. We also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells.

Download Full-text

Monoclonal Antibody 667 Recognizes the Variable Region A Motif of the Ecotropic Retrovirus CasBrE Envelope Glycoprotein and Inhibits Env Binding to the Viral Receptor

Journal of Virology ◽

10.1128/jvi.77.20.10984-10993.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 10984-10993 ◽

Cited By ~ 8

Author(s):

Hanna Dreja ◽

Laurent Gros ◽

Sylvie Villard ◽

Estanislao Bachrach ◽

Anna Oates ◽

...

Keyword(s):

Amino Acids ◽

Monoclonal Antibody ◽

Receptor Binding ◽

Envelope Glycoprotein ◽

Critical Role ◽

Variable Region ◽

Binding Domain ◽

Target Cells ◽

Kinetic Properties ◽

Receptor Binding Domain

ABSTRACT Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D57, is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

Download Full-text

Effect of RBD (Y453F) mutation in spike glycoprotein of SARS-CoV-2 on neutralizing IgG affinity

10.1101/2021.01.28.21250577 ◽

2021 ◽

Author(s):

Takuma Hayashi ◽

Nobuo Yaegashi ◽

Ikuo Konishi

Keyword(s):

Monoclonal Antibodies ◽

Structural Analysis ◽

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Virus Disease ◽

Three Dimensional ◽

Binding Domain ◽

Receptor Binding Domain ◽

Spike Glycoprotein

AbstractBackgroundInfection with receptor binding domain (RBD) mutant (Y453F) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from farmed minks is known to widely spread among humans.MethodsWe investigated the characteristics of SARS-CoV-2 RBD Y453F mutant using three- dimensional structural analysis. We investigated the effect of the RBD Y453F mutant of SARS-CoV- 2 on neutralizing antibodies in serum derived from Corona virus Disease 2019 (COVID-19) positive patients.ResultsOur studies suggest that virus variants with RBD Y453F mutation partially escaped detection by four neutralizing monoclonal antibodies and neutralizing antibodies in serum.ConclusionsConsequently, raising a concern that infection of SARS-CoV-2 mutants that cause serious symptoms in humans may spread globally.

Download Full-text

Molecular Docking study of Receptor Binding Domain of SARS-CoV-2 Spike Glycoprotein with Saikosaponin, a Triterpenoid Natural Product

10.26434/chemrxiv.12033774.v1 ◽

2020 ◽

Cited By ~ 2

Author(s):

Tamal Goswami ◽

Bhaskar Bagchi

Keyword(s):

Molecular Docking ◽

Receptor Binding ◽

Docking Study ◽

Binding Domain ◽

Target Cells ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Molecular Docking Study ◽

Computational Docking ◽

Angiotensin Converting Enzyme 2

The appearance of SARS-CoV-2 has resulted ~19000 deaths and ~423000 infections worldwide as of March 24, 2020. Coronavirus spike (S) glycoproteins hooks on target cells and binds to the angiotensin-converting enzyme 2 (ACE2) receptor. Recent researches speculated that residues 331 to 524 of the S glycoprotein of the receptor binding domain (RDB) of the spike is the most crucial target and this side was very important for computational docking. In the present study we have considered a series of saikosaponins and molecular docking was performed. Most of the docked molecules bind favorably to the RDB region of the spike glycoprotein and among them Saikosaponin B4 is the best inhibitor.

Download Full-text

Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

mBio ◽

10.1128/mbio.00862-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 60

Author(s):

Nicholas J. Lennemann ◽

Bethany A. Rhein ◽

Esther Ndungo ◽

Kartik Chandran ◽

Xiangguo Qiu ◽

...

Keyword(s):

Receptor Binding ◽

Ebola Virus ◽

Binding Domain ◽

Marburg Virus ◽

Viral Envelope ◽

Targeted Mutagenesis ◽

Site Directed Mutagenesis ◽

Receptor Binding Domain ◽

Antibody Neutralization ◽

Glycosylation Sites

ABSTRACTEbola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca2+-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency.IMPORTANCEFilovirus outbreaks occur sporadically throughout central Africa, causing high fatality rates among the general public and health care workers. These unpredictable hemorrhagic fever outbreaks are caused by multiple species of Ebola viruses, as well as Marburg virus. While filovirus vaccines and therapeutics are being developed, there are no licensed products. The sole viral envelope glycoprotein, which is a principal immunogenic target, contains a heavy shield of glycans surrounding the conserved receptor-binding domain. We find that disruption of this shield through targeted mutagenesis leads to an increase in cell entry, protease sensitivity, and antiserum/antibody sensitivity but is not sufficient to allow virion binding to the intracellular receptor NPC1. Therefore, our studies provide evidence that filoviruses maintain glycoprotein glycosylation to protect against proteases and antibody neutralization at the expense of efficient entry. Our results unveil interesting insights into the unique entry process of filoviruses and potential immune evasion tactics of the virus.

Download Full-text

Characterization of the Prototype Foamy Virus Envelope Glycoprotein Receptor-Binding Domain

Journal of Virology ◽

10.1128/jvi.00460-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 8158-8167 ◽

Cited By ~ 11

Author(s):

Anja Duda ◽

Daniel Lüftenegger ◽

Thomas Pietschmann ◽

Dirk Lindemann

Keyword(s):

Host Cell ◽

Receptor Binding ◽

Foamy Virus ◽

Glycosylation Site ◽

Binding Domain ◽

Leader Peptide ◽

Target Cells ◽

Receptor Binding Domain ◽

Cell Binding ◽

Extracellular Domains

ABSTRACT The foamy virus (FV) glycoprotein precursor gp130Env undergoes a highly unusual biosynthesis, resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM). Little structural and functional information on the extracellular domains of FV Env is available. In this study, we characterized the prototype FV (PFV) Env receptor-binding domain (RBD) by flow cytometric analysis of recombinant PFV Env immunoadhesin binding to target cells. The extracellular domains of the C-terminal TM subunit as well as targeting of the recombinant immunoadhesins by the cognate LP to the secretory pathway were dispensable for target cell binding, suggesting that the PFV Env RBD is contained within the SU subunit. N- and C-terminal deletion analysis of the SU domain revealed a minimal continuous RBD spanning amino acids (aa) 225 to 555; however, internal deletions covering the region from aa 397 to 483, but not aa 262 to 300 or aa 342 to 396, were tolerated without significant influence on host cell binding. Analysis of individual cysteine point mutants in PFV SU revealed that only most of those located in the nonessential region from aa 397 to 483 retained residual binding activity. Interestingly, analysis of various N-glycosylation site mutants suggests an important role of carbohydrate chain attachment to N391, either for direct interaction with the receptor or for correct folding of the PFV Env RBD. Taken together, these results suggest that a bipartite sequence motif spanning aa 225 to 396 and aa 484 to 555 is essential for formation of the PFV Env RBD, with N-glycosylation site at position 391 playing a crucial role for host cell binding.

Download Full-text

Infectivity and antigenicity of SARS-CoV-2 B.1.617 variants

10.21203/rs.3.rs-596463/v1 ◽

2021 ◽

Author(s):

Youchun Wang ◽

Li Zhang ◽

Qianqian Li ◽

Jiajing Wu ◽

Yuanling Yu ◽

...

Keyword(s):

Amino Acid ◽

Cell Fusion ◽

Receptor Binding ◽

Human Cells ◽

Binding Domain ◽

Single Amino Acid ◽

Inactivated Vaccine ◽

Receptor Binding Domain ◽

Amino Acid Mutations ◽

Cell Cell

Abstract SARS-CoV-2 has caused the COVID-19 pandemic. Recently, B.1.617 variants have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell–cell fusion after infection of B.1.617 variants was enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.

Download Full-text

Coronaviruses with a SARS-CoV-2-like receptor-binding domain allowing ACE2-mediated entry into human cells isolated from bats of Indochinese peninsula

10.21203/rs.3.rs-871965/v1 ◽

2021 ◽

Author(s):

Sarah Temmam ◽

Khamsing Vongphayloth ◽

Eduard Baquero Salazar ◽

Sandie Munier ◽

Max Bonomi ◽

...

Keyword(s):

Receptor Binding ◽

Human Cells ◽

Binding Domain ◽

Receptor Binding Domain ◽

Furin Cleavage ◽

Animal Reservoir ◽

Angiotensin Converting Enzyme 2 ◽

Furin Cleavage Site ◽

Spike Sequence ◽

Early Human

Abstract The animal reservoir of SARS-CoV-2 is unknown despite reports of various SARS-CoV-2-related viruses in Asian Rhinolophus bats, including the closest virus from R. affinis, RaTG13. Several studies have suggested the involvement of pangolin coronaviruses in SARS-CoV-2 emergence. SARS-CoV-2 presents a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain (RBD) to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 pathway have not yet been identified, though they would be key in understanding the origin of the epidemics. Here we show that such viruses indeed circulate in cave bats living in the limestone karstic terrain in North Laos, within the Indochinese peninsula. We found that the RBDs of these viruses differ from that of SARS-CoV-2 by only one or two residues, bind as efficiently to the hACE2 protein as the SARS-CoV-2 Wuhan strain isolated in early human cases, and mediate hACE2-dependent entry into human cells, which is inhibited by antibodies neutralizing SARS-CoV-2. None of these bat viruses harbors a furin cleavage site in the spike. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.

Download Full-text