scholarly journals Substitutions at the Putative Receptor-Binding Site of an Encephalitic Flavivirus Alter Virulence and Host Cell Tropism and Reveal a Role for Glycosaminoglycans in Entry

2000 ◽  
Vol 74 (19) ◽  
pp. 8867-8875 ◽  
Author(s):  
Eva Lee ◽  
Mario Lobigs
Keyword(s):  
Host Cell ◽  
Receptor Binding ◽  
Mammalian Cells ◽  
Virus Entry ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Virus Infectivity ◽  
Cell Tropism ◽  
E Protein ◽  
The Impact

ABSTRACT The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp390, which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp390 on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly390, Ala390, and His390) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

scholarly journals Design of a companion bioinformatic tool to detect the emergence and geographical distribution of SARS-CoV-2 Spike protein genetic variants

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Alice Massacci ◽  
Eleonora Sperandio ◽  
Lorenzo D’Ambrosio ◽  
Mariano Maffei ◽  
Fabio Palombo ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Consensus Sequence ◽  
Cell Epitope ◽  
Vaccine Design ◽  
Binding Domain ◽  
Spike Protein ◽  
Antibody Activity ◽  
Binding Motif ◽  
Receptor Binding Domain ◽  
The Impact

Abstract Background Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. Methods Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. Results Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. Conclusions This work provides a framework able to track down SARS-CoV-2 genomic variability.

scholarly journals Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies

2021 ◽  
Author(s):  
Allison J. Greaney ◽  
Andrea N. Loes ◽  
Katharine H.D. Crawford ◽  
Tyler N. Starr ◽  
Keara D. Malone ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Domain ◽  
Binding Motif ◽  
Human Antibody ◽  
Receptor Binding Domain ◽  
Serum Antibodies ◽  
Main Target ◽  
The Impact ◽  
Polyclonal Serum ◽  
Comprehensive Mapping

AbstractThe evolution of SARS-CoV-2 could impair recognition of the virus by human antibody-mediated immunity. To facilitate prospective surveillance for such evolution, we map how convalescent serum antibodies are impacted by all mutations to the spike’s receptor-binding domain (RBD), the main target of serum neutralizing activity. Binding by polyclonal serum antibodies is affected by mutations in three main epitopes in the RBD, but there is substantial variation in the impact of mutations both among individuals and within the same individual over time. Despite this inter- and intra-person heterogeneity, the mutations that most reduce antibody binding usually occur at just a few sites in the RBD’s receptor binding motif. The most important site is E484, where neutralization by some sera is reduced >10-fold by several mutations, including one in emerging viral lineages in South Africa and Brazil. Going forward, these serum escape maps can inform surveillance of SARS-CoV-2 evolution.

scholarly journals Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
James A. Williams ◽  
Long Gui ◽  
Nancy Hom ◽  
Alexander Mileant ◽  
Kelly K. Lee
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Virus Infectivity ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Influenza Virus Hemagglutinin ◽  
Fusion Glycoprotein ◽  
The Impact

ABSTRACTThe neutralizing antibody (nAb) response against the influenza virus hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency in the inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor binding pocket at the distal end of HA, while FI6v3 binds primarily to the HA2 fusion subunit toward the base of the stalk. Surprisingly, HC19 inhibited the ability of HA to induce lipid mixing by preventing the structural rearrangement of HA under fusion-activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to cross-link separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab fragment was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that interspike cross-linking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared-down Fab-soluble HA structures.IMPORTANCEThe influenza virus hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of antibody (Ab)-mediated neutralization largely relies on the high-resolution characterization of antigen binding (Fab) fragments in complex with soluble, isolated antigen constructs by cryo-electron microscopy (EM) single-particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occur by multiple coexisting mechanisms, are largely dependent on the specific epitope that is targeted, and are highly dependent on the bivalent nature of IgG molecules.

scholarly journals Expansion of host-cell tropism of foot-and-mouth disease virus despite replication in a constant environment

2004 ◽  
Vol 85 (8) ◽  
pp. 2289-2297 ◽  
Author(s):  
Carmen M. Ruiz-Jarabo ◽  
Nonia Pariente ◽  
Eric Baranowski ◽  
Mercedes Dávila ◽  
Gema Gómez-Mariano ◽  
...  
Keyword(s):  
Host Cell ◽  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Jurkat Cells ◽  
Virus Infectivity ◽  
Cell Tropism ◽  
Cellular Environment ◽  
Mouth Disease Virus ◽  
Foot And Mouth

Foot-and-mouth disease virus (FMDV) variants adapted to BHK-21 cells showed an expanded host-cell tropism that extended to primate and human cell lines. Virus replication in human HeLa and Jurkat cells has been documented by titration of virus infectivity, quantification of virus RNA, expression of a virus-specific non-structural antigen, and serial passage of virus in the cells. Parallel serial infections of human Jurkat cells with the same variant FMDVs indicates a strong stochastic component in the progression of infection. Chimeric viruses identified the capsid as a genomic region involved in tropism expansion. These results indicate that, contrary to theoretical predictions, replication of an RNA virus in a constant cellular environment may lead to expansion of cellular tropism, rather than to a more specialized infection of the cellular type to which the virus has been adapted.

scholarly journals Spike Protein of SARS-CoV-2: Impact of Single Amino Acid Mutation and Effect of Drug Binding to the Variant-in Silico Analysis

2020 ◽  
Author(s):  
Pratibha Manickavasagam
Keyword(s):  
Amino Acid ◽  
Host Cell ◽  
Drug Binding ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
S Protein ◽  
The Impact ◽  
Single Amino Acid Mutation

Novel SARS-CoV-2, a bat based virus originated in Wuhan, China that caused a global pandemic in December, 2019 belongs to the Betacorona virus family and contains single stranded genome of ~29Kbp. The host cell invasion of SARS-CoV-2 is facilitated by interaction of C-Terminal Domain (CTD) of Spike (S) protein of virus and host ACE2 receptor in the presence of TMPRSS seine protease secreted by the host cell. In this study the mutation hotspots of S-protein will be identified and the impact of such mutation in the binding affinity will be studied. Additionally, the lead molecule which can bind to the mutated protein also will be identified. Multiple sequence alignment of the spike protein sequence of SARS-CoV-2 shows the number of single amino acid mutation hotspots such as L5F, R214L, R408I, G476S, V483A, H519Q, A520S, T572I, D614G and H655Y. Among these mutations D614G has 57.5% occurrence and G476S, V483A has 7.5% occurrence. The mutated proteins were modelled based on wild type homolog and docked to ACE2 receptor. When the mutated S protein is docked, the ∆G (binding free energy) value is very minimal in mutated protein showed the stability of variants. By the drug repurposing method, 1000 FDA approved drugs were virtually screened for its binding to RBD of S1 domain. Among these drugs Digitoxin, Gliquidone and Zorubicin Hcl binds to spike proteins with higher docking score (lesser than -8.5 Kcal/mol) to both wild type and mutants.

scholarly journals Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif

2021 ◽  
Author(s):  
Ramiro Lorenzo ◽  
Lucas A. Defelipe ◽  
Lucio Aliperti ◽  
Stephan Niebling ◽  
Tânia F. Custódio ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Strong Dependence ◽  
Computational Prediction ◽  
Binding Motif ◽  
Virus Infectivity ◽  
Asparagine Deamidation ◽  
Molecular Aging ◽  
Virion Surface

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 °C, respectively. This process is significantly slowed down at 4 °C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.

scholarly journals Attenuation of Tick-Borne Encephalitis Virus by Structure-Based Site-Specific Mutagenesis of a Putative Flavivirus Receptor Binding Site

2000 ◽  
Vol 74 (20) ◽  
pp. 9601-9609 ◽  
Author(s):  
Christian W. Mandl ◽  
Steven L. Allison ◽  
Heidemarie Holzmann ◽  
Tamara Meixner ◽  
Franz X. Heinz
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Biological Effects ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Dimensional Structure ◽  
Virus Protein ◽  
Tick Borne Encephalitis ◽  
Tbe Virus ◽  
The Impact

ABSTRACT The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3′-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.

scholarly journals Influenza H1N1 A/Solomon Island/3/06 Virus Receptor Binding Specificity Correlates with Virus Pathogenicity, Antigenicity, and Immunogenicity in Ferrets

2010 ◽  
Vol 84 (10) ◽  
pp. 4936-4945 ◽  
Author(s):  
Qi Xu ◽  
Weijia Wang ◽  
Xing Cheng ◽  
James Zengel ◽  
Hong Jin
Keyword(s):  
Sialic Acid ◽  
Respiratory Tract ◽  
Receptor Binding ◽  
Lower Respiratory Tract ◽  
Solomon Island ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Wild Type ◽  
Single Amino Acid Change ◽  
The Impact

ABSTRACT Influenza viruses attach to cells via a sialic acid moiety (sialic acid receptor) that is α2-3 linked or α2-6 linked to galactose (α2-3SAL or α2-6SAL); sialic acid acts as a receptor for the virus. Using lectin staining, we demonstrated that the α2-6SAL configuration is predominant in the respiratory tract of ferrets, including trachea, bronchus, and lung alveolus tissues. Recombinant wild-type (rWT) influenza A/Solomon Island/3/06 (SI06) (H1N1) viruses were constructed to assess the impact of the hemagglutinin (HA) variations (amino acids 190 or 226) identified in natural variants on virus replication in the upper and lower respiratory tract of ferrets, as well as virus antigenicity and immunogenicity. A single amino acid change at residue 226 (from Gln to Arg) in the HA of SI06 resulted in the complete loss of binding to α2-6SAL and a concomitant loss of the virus's ability to replicate in the lower respiratory tract of ferrets. In contrast, the virus with Gln226 in the HA protein has a receptor binding preference for α2-6SAL and replicates efficiently in the lungs. There was a good correlation between viral replication in the lungs of ferrets and disease symptoms. In addition, we also showed that the 190 and 226 residues affected viral antigenicity and immunogenicity. Our data emphasize the necessity of thoroughly assessing wild-type influenza viruses for their suitability as reference strains and for carefully selecting the HA antigen for vaccine production during annual influenza vaccine evaluation processes.

scholarly journals A human ex vivo dengue virus neutralization assay identifies priority antibodies and epitopes for vaccines and therapeutics

2019 ◽  
Author(s):  
Trung Tuan Vu ◽  
Hannah Clapham ◽  
Van Thi Thuy Huynh ◽  
Long Vo Thi ◽  
Dui Le Thi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Dengue Virus ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Virus Infectivity ◽  
Virus Neutralization Assay ◽  
The Impact ◽  
Arboviral Disease

AbstractBackgroundDengue is the most prevalent arboviral disease, for which neither effective vaccines nor antivirals are available. Clinical trials with Dengvaxia, the first licensed dengue vaccine, show the conventional in vitro plaque reduction neutralization test (PRNT) failed to discriminate between neutralizing and non-neutralizing antibodies. A number of human monoclonal antibodies (mAbs) were characterized by PRNT as being neutralizers of virus infectivity for mammalian cells.Methodolody/Principle findingsWe developed a neutralization assay and tested the capacity of 12 mAbs to neutralize the infectiousness of dengue patient viremic blood in mosquitoes. We identified minimum concentrations of a subset of mAbs required to achieve dengue virus neutralization, and modelled the impact of a therapeutic mAb candidate on viremia.Five of the 12 mAbs (14c10, 2D22, 1L12, 747(4)B7, 753(3)C10), all of which target quaternary epitopes, potently inhibited dengue virus infection of Ae. aegypti. The potency of several mAbs was compromised in the context of patients with secondary serological profiles, possibly reflecting competition between the exogenously-added mAbs and the patient’s own antibody responses at or near the target epitopes. The minimum concentrations that mAbs neutralized DENV ranged from 0.1 – 5 µg/mL. An Fc-disabled variant of mAb (14c10-LALA) was as potent as its parent mAb. Within-host mathematical modelling suggests infusion of 14c10-LALA could bring about rapid acceleration of viremia resolution in a typical patient.Conclusions/SignificanceThese data delivered a unique assessment of anti-viral potency of a panel of human mAbs. Results support the advancement of dengue virus neutralization assays, and the development of therapeutics against flaviviruses, to which dengue virus and Zika virus belong.Author summaryDengue is the most prevalent arboviral disease affecting humans. There are no therapeutics for the disease. Antibody-mediated immunity against dengue is also not well-understood, as shown by the failure of the conventional neutralization assay used to predict the efficacy of Dengvaxia, the first licensed vaccine for the disease. It is likely that the neutralization assay targets non-neutralizing antibodies, but there are no validation assays available. To this end, we developed a novel virus neutralization assay, employing Aedes aegypti mosquitoes and viremic blood from dengue patients, to examine the virus-neutralizing potency of 12 human-derived monoclonal antibodies (mAbs). While all of these mAbs neutralized dengue virus using the conventional assay, seven of them failed to block dengue virus infections of mosquitoes using our assay. The remaining five mAbs neutralized at least one serotype of dengue virus and the minimum neutralizing concentrations of range from 0.1 – 5 µg/mL. Using the minimum neutralizing concentration of a therapeutic mAb candidate, we investigated the impact of the mAb on viremia using a mathematical model and found the mAb accelerated the reduction of viremia. The results support the advancement of dengue virus neutralization assays, and the development of therapeutics for dengue.

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