Glycan Receptor Binding Research Articles

Patterns of the within-host evolution of human norovirus in immunocompromised individuals and implications for treatment

Currently, there is no licensed treatment for chronic norovirus infections, but the use of intra-duodenally-delivered immunoglobulins is promising; nevertheless, varying results have limited their wide use. Little is known about the relationship between norovirus genetic diversity and treatment efficacy. We analyzed the norovirus within-host diversity and evolution in a cohort of 20 immunocompromised individuals using next-generation sequencing (NGS) and clone-based sequencing of the capsid (VP1) gene. Representative VP1s were expressed and their glycan receptor binding affinity and antigenicity were evaluated. The P2 domain, within the VP1, accumulated up to 30-fold more non-synonymous mutations than other genomic regions. Intra-host virus populations in these patients tended to evolve into divergent lineages that were often antigenically distinct. Several of these viruses were widely resistant to binding-blocking antibodies in immunoglobulin preparations. Notably, for one patient, a single amino-acid substitution in the P2 domain resulted in an immune-escape phenotype, and it was likely the main contributor to treatment failure. Furthermore, we found evidence for transmission of late-stage viruses between two immunocompromised individuals. The findings demonstrated that within-host noroviruses in chronic infections tend to evolve into antigenically distinct subpopulations. This antigenic evolution was likely caused by the remaining low immunity levels exerted by immunocompromised individuals, possibly undermining antiviral treatment. Our observations provide insights into norovirus (within-host) evolution and treatment. Erasmus MC grant mRACE, the European Union's Horizon 2020 research and innovation program under grant agreement No. 874735 (VEO), and the NWO STEVIN award (Koopmans).

A Pseudovirus Nanoparticle-Based Trivalent Rotavirus Vaccine Candidate Elicits High and Cross P Type Immune Response.

Rotavirus infection continues to cause significant morbidity and mortality globally. In this study, we further developed the S60-VP8* pseudovirus nanoparticles (PVNPs) displaying the glycan receptor binding VP8* domains of rotavirus spike proteins as a parenteral vaccine candidate. First, we established a scalable method for the large production of tag-free S60-VP8* PVNPs representing four rotavirus P types, P[8], P[4], P[6], and P[11]. The approach consists of two major steps: selective precipitation of the S-VP8* proteins from bacterial lysates using ammonium sulfate, followed by anion exchange chromatography to further purify the target proteins to a high purity. The purified soluble proteins self-assembled into S60-VP8* PVNPs. Importantly, after intramuscular injections, the trivalent vaccine consisting of three PVNPs covering VP8* antigens of P[8], P[4], and P[6] rotaviruses elicited high and broad immunogenicity in mice toward the three predominant P-type rotaviruses. Specifically, the trivalent vaccine-immunized mouse sera showed (1) high and balanced IgG and IgA antibody titers toward all three VP8* types, (2) high blocking titer against the VP8*-glycan receptor interaction, and (3) high and broad neutralizing titers against replications of all P[8], P[4], and P[6] rotaviruses. Therefore, trivalent S60-VP8* PVNPs are a promising non-replicating, parenteral vaccine candidate against the most prevalent rotaviruses worldwide.

The structural basis of Salmonella A2B5 toxin neutralization by antibodies targeting the glycan-receptor binding subunits.

SUMMARYMany bacterial pathogens secrete A(2)B5 toxins comprising two functionally distinct yet complementary “A” and “B” subunits to benefit the pathogens during infection. The lectin-like pentameric B subunits recognize specific sets of host glycans to deliver the toxin into target host cells. Here, we offer the molecular mechanism by which neutralizing antibodies, which have the potential to bind to all glycan-receptor binding sites and thus completely inhibit toxin binding to host cells, are inhibited from exerting this action. Cryogenic electron microscopy (cryo-EM)-based analyses indicate that the skewed positioning of the toxin A subunit(s) toward one side of the toxin B pentamer inhibited neutralizing antibody binding to the laterally located epitopes, rendering some glycan-receptor binding sites that remained available for the toxin binding and endocytosis process, which is strikingly different from the counterpart antibodies recognizing the far side-located epitopes. These results highlight additional features of the toxin-antibody interactions and offer important insights into anti-toxin strategies.

Mechanisms of typhoid toxin neutralization by antibodies targeting glycan receptor binding and nuclease subunits

SummaryNearly all clinical isolates of Salmonella Typhi, the cause of typhoid fever, are antibiotic resistant. All S. Typhi isolates secrete an A2B5 exotoxin called typhoid toxin to benefit the pathogen during infection. Here, we demonstrate that antibiotic-resistant S. Typhi secretes typhoid toxin continuously during infection regardless of antibiotic treatment. We characterize typhoid toxin antibodies targeting glycan-receptor-binding PltB or nuclease CdtB, which neutralize typhoid toxin in vitro and in vivo, as demonstrated by using typhoid toxin secreted by antibiotic-resistant S. Typhi during human cell infection and lethal dose typhoid toxin challenge to mice. TyTx11 generated in this study neutralizes typhoid toxin effectively, comparable to TyTx4 that binds to all PltB subunits available per holotoxin. Cryoelectron microscopy explains that the binding of TyTx11 to CdtB makes this subunit inactive through CdtB catalytic-site conformational change. The identified toxin-neutralizing epitopes are conserved across all S. Typhi clinical isolates, offering critical insights into typhoid toxin-neutralizing strategies.

A Nanoparticle-Based Trivalent Vaccine Targeting the Glycan Binding VP8* Domains of Rotaviruses.

Rotavirus causes severe gastroenteritis in children. Although vaccines are implemented, rotavirus-related diarrhea still claims ~200,000 lives annually worldwide, mainly in low-income settings, pointing to a need for improved vaccine tactics. To meet such a public health need, a P24-VP8* nanoparticle displaying the glycan-binding VP8* domains, the major neutralizing antigens of rotavirus, was generated as a new type of rotavirus vaccine. We reported here our development of a P24-VP8* nanoparticle-based trivalent vaccine. First, we established a method to produce tag-free P24-VP8* nanoparticles presenting the VP8*s of P[8], P[4], and P[6] rotaviruses, respectively, which are the three predominantly circulating rotavirus P types globally. This approach consists of a chemical-based protein precipitation and an ion exchange purification, which may be scaled up for large vaccine production. All three P24-VP8* nanoparticle types self-assembled efficiently with authentic VP8*-glycan receptor binding function. After they were mixed as a trivalent vaccine, we showed that intramuscular immunization of the vaccine elicited high IgG titers specific to the three homologous VP8* types in mice. The resulted mouse sera strongly neutralized replication of all three rotavirus P types in cell culture. Thus, the trivalent P24-VP8* nanoparticles are a promising vaccine candidate for parenteral use against multiple P types of predominant rotaviruses.

Structural basis for Glycan-receptor binding by mumps virus hemagglutinin-neuraminidase

Mumps virus is one of the main cause of respiratory illnesses in humans, especially children. Among the viral surface glycoproteins, the hemagglutinin – neuraminidase, MuV-HN, plays key roles in virus entry into host cells and infectivity, thus representing an ideal target for the design of novel inhibitors. Here we report the detailed analysis of the molecular recognition of host cell surface sialylated glycans by the viral glycoprotein MuV-HN. By a combined use of NMR, docking, molecular modelling and CORCEMA-ST, the structural features of sialoglycans/MuV-HN complexes were revealed. Evidence for a different enzyme activity toward longer and complex substrates compared to unbranched ligands was also examined by an accurate NMR kinetic analysis. Our results provide the basis for the structure-based design of effective drugs against mumps-induced diseases.

Disruption of the Dimer-Dimer Interaction of the Mumps Virus Attachment Protein Head Domain, Aided by an Anion Located at the Interface, Compromises Membrane Fusion Triggering.

Mumps virus (MuV), an enveloped negative-strand RNA virus belonging to the family Paramyxoviridae, enters the host cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin-neuraminidase (MuV-HN) and a fusion (F) protein. However, how the binding of MuV-HN to glycan receptors triggers membrane fusion is not well understood. The crystal structure of the MuV-HN head domain forms a tetramer (dimer of dimers) like other paramyxovirus attachment proteins. In the structure, a sulfate ion (SO42-) was found at the interface between two dimers, which may be replaced by a hydrogen phosphate ion (HPO42-) under physiological conditions. The anion is captured by the side chain of a positively charged arginine residue at position 139 of one monomer each from both dimers. Substitution of alanine or lysine for arginine at this position compromised the fusion support activity of MuV-HN without affecting its cell surface expression, glycan-receptor binding, and interaction with the F protein. Furthermore, the substitution appeared to affect the tetramer formation of the head domain as revealed by blue native-PAGE analysis. These results, together with our previous similar findings with the measles virus attachment protein head domain, suggest that the dimer-dimer interaction within the tetramer may play an important role in triggering membrane fusion during paramyxovirus entry.IMPORTANCE Despite the use of effective live vaccines, mumps outbreaks still occur worldwide. Mumps virus (MuV) infection typically causes flu-like symptoms and parotid gland swelling but sometimes leads to orchitis, oophoritis, and neurological complications, such as meningitis, encephalitis, and deafness. MuV enters the host cell through membrane fusion mediated by two viral proteins, a receptor-binding attachment protein, and a fusion protein, but its detailed mechanism is not fully understood. In this study, we show that the tetramer (dimer of dimers) formation of the MuV attachment protein head domain is supported by an anion located at the interface between two dimers and that the dimer-dimer interaction plays an important role in triggering the activation of the fusion protein and causing membrane fusion. These results not only further our understanding of MuV entry but provide useful information about a possible target for antiviral drugs.

Host Adaptations of the <i>Salmonella</i> Typhi Typhoid Toxin and Its Orthologue from a Nontyphoidal <i>Salmonella</i>

Typhoidal and nontyphoidal Salmonellae (NTS) cause typhoid fever and gastroenteritis respectively in humans. Typhoid toxin from typhoidal Salmonella contributes to typhoid disease progression and chronic infection, but little is known about its orthologue from NTS. We found that these two Salmonella toxins do not cause similar toxicities in vivo. The altered virulence outcomes are due to the altered glycan binding preferences of these two toxins, which are correlated to the glycan expression profiles of target host cells at the primary infection or intoxication sites of their associated bacteria. The co-crystal structures of the glycan-receptor binding PltB subunits of the toxins bound to specific glycan receptor moieties explain how PltBs of these two toxins display markedly different glycan binding preferences and contribute to the subsequent virulence outcomes. We also revealed that administration of either Javiana toxin or S. Javiana into an animal model provides cross-reactive protection for typhoid-related illnesses and mortality.

The bulky and the sweet: How neutralizing antibodies and glycan receptors compete for virus binding.

Numerous viruses rely on glycan receptor binding as the initial step in host cell infection. Engagement of specific glycan receptors such as sialylated carbohydrates, glycosaminoglycans, or histo‐blood group antigens can determine host range, tissue tropism, and pathogenicity. Glycan receptor‐binding sites are typically located in exposed regions on viral surfaces—sites that are also generally prone to binding of neutralizing antibodies that directly interfere with virus‐glycan receptor interactions. In this review, we examine the locations and architecture of the glycan‐ and antibody‐binding sites in four different viruses with stalk‐like attachment proteins (reovirus, influenza virus, norovirus, and coronavirus) and investigate the mechanisms by which antibodies block glycan recognition. Those viruses exemplify that direct molecular mimicking of glycan receptors by antibodies is rare and further demonstrate that antibodies often partly overlap or bind sufficiently close to the receptor‐binding region to hinder access to this site, achieving neutralization partially because of the epitope location and partly due to their sheer size.

Was ist Glykovirologie?

Cell-surface glycans play central roles in mediating the cell attachment and entry of many viruses, including a number of human pathogens. However, glycans are notoriously difficult to study. The emerging field of “glycovirology” aims to provide direct insight into biophysical mechanisms that determine viral tropism, to correlate glycan receptor binding with pathogenicity, and to ultimately provide a platform from which novel glycan-based antiviral strategies can be launched.

Substantial Receptor-induced Structural Rearrangement of Rotavirus VP8*: Potential Implications for Cross-Species Infection.

Rotavirus-cell binding is the essential first step in rotavirus infection. This binding is a major determinant of rotavirus tropism, as host cell invasion is necessary to initiate infection. Initial rotavirus-cell interactions are mediated by carbohydrate-recognizing domain VP8* of the rotavirus capsid spike protein VP4. Here, we report the first observation of significant structural rearrangement of VP8* from human and animal rotavirus strains upon glycan receptor binding. The structural adaptability of rotavirus VP8* delivers important insights into how human and animal rotaviruses utilize the wider range of cellular glycans identified as VP8* binding partners. Furthermore, our studies on rotaviruses with atypical genetic makeup provide information expected to be critical for understanding the mechanisms of animal rotavirus gene emergence in humans and support implementation of epidemiologic surveillance of animal reservoirs as well as future vaccination schemes.

Insights into the human glycan receptor conformation of 1918 pandemic hemagglutinin-glycan complexes derived from nuclear magnetic resonance and molecular dynamics studies.

The glycan receptor binding and specificity of influenza A viral hemagglutinin (HA) are critical for virus infection and transmission in humans. However, ambiguities in the interpretation of the receptor binding specificity of hemagglutinin from human- and avian-adapted viruses have prevented an understanding of its relationship with aerosol transmissibility, an exclusive property of human-adapted viruses. A previous conformational study, which we performed, indicated that human and avian receptors sample distinct conformations in solution. On the basis of detailed nuclear magnetic resonance (NMR) studies provided herein, we offer evidence of the distinct structural constraints imposed by hemagglutinin receptor binding sites on the glycan conformational space upon binding. The hemagglutinin from the SC18 virus, which has efficient aerosol transmissibility in humans (human-adapted), imposed the most stringent constraints on the conformational space of the human glycan receptor (LSTc), compared to single (NY18) or double (AV18) amino acid HA mutants, a property correlating to the ligand–HA binding strength. This relationship was also observed for the avian-adapted HA, where the high affinity binding partner, AV18, imposed the most stringent conformational constraints on the avian receptor, compared to those imposed by NY18. In particular, it is interesting to observe how different HAs when binding to human or avian glycosidic receptors impose significantly different conformational states, in terms of the states sampled by the glycosidic backbone and/or the entire molecule shape (linear or bent), when compared to the corresponding unbound glycans. Significantly, we delineate a “characteristic NMR signature” for the human adapted hemagglutinin (SC18) binding to human glycan receptors. Therefore, the conformational space constraints imposed by the hemagglutinin receptor binding site provide a characteristic signature that could be a useful tool for the surveillance of human adaptation of other (such as H7N9 and H5N1) deadly influenza viruses.

N-Linked Glycosylation of the Hemagglutinin Protein Influences Virulence and Antigenicity of the 1918 Pandemic and Seasonal H1N1 Influenza A Viruses

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.

Quantitative Characterization of Glycan-Receptor Binding of H9N2 Influenza A Virus Hemagglutinin

Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA) and neuraminidase (NA) genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1) subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.

Glycan Binding Avidity Determines the Systemic Fate of Adeno-Associated Virus Type 9

Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions.

Molecular mechanism of the enhanced virulence of 2009 pandemic Influenza A (H1N1) virus from D222G mutation in the hemagglutinin: a molecular modeling study

D222G mutation of the hemagglutinin (HA) is of special interest because of its close association with the enhanced virulence of 2009 pandemic influenza A (H1N1) virus through the increased binding affinity to α2,3-linked sialylated glycan receptors. However, there is still a lack of detailed understanding about the molecular mechanism of this enhanced virulence. Here, molecular dynamics simulation and binding free energy calculation were performed to explore the altered glycan receptor binding mechanism of HA upon the D222G mutation by studying the interaction of one α2,3-linked sialylglycan (sequence: SIA-GAL-NAG) with the wild type and D222G mutated HA. The binding free energy calculation based on the molecular mechanics generalized Born surface area (MM-GBSA) method indicates that the D222G mutated HA has a much stronger binding affinity with the studied α2,3-linked glycan than the wild type. This is consistent with the experimental result. The increased binding free energy of D222G mutant mainly comes from the increased energy contribution of Gln223. The structural analysis proves that the altered electrostatic potential of receptor binding domain (RBD) and the increased flexibility of 220-loop are the essential reasons leading to the increased affinity of HA to α2,3-linked sialic acid glycans. The obtained results of this study have allowed a deeper understanding of the receptor recognition mechanism and the pathogenicity of influenza virus, which will be valuable to the structure-based inhibitors design targeting influenza virus entry process.

Glycomics‐based analysis of chicken red blood cells provides insight into the selectivity of the viral agglutination assay

Agglutination of red blood cells (RBCs), including chicken RBCs (cRBCs), has been used extensively to estimate viral titer, to screen glycan-receptor binding preference, and to assess the protective response of vaccines. Although this assay enjoys widespread use, some virus strains do not agglutinate RBCs. To address these underlying issues and to increase the usefulness of cRBCs as tools for studying viruses, such as influenza, we analyzed the cell surface N-glycans of cRBCs. On the basis of the results obtained from complementary analytical strategies, including MS, 1D and 2D-NMR spectroscopy, exoglycosidase digestions, and HPLC profiling, we report the major glycan structures present on cRBCs. By comparing the glycan structures of cBRCs with those of representative human upper respiratory cells, we offer a possible explanation for the fact that certain influenza strains do not agglutinate cRBCs, using specific human-adapted influenza hemagglutinins as examples. Finally, recent understanding of the role of various glycan structures in high affinity binding to influenza hemagglutinins provides context to our findings. These results illustrate that the field of glycomics can provide important information with respect to the experimental systems used to characterize, detect and study viruses.

Determinants of Glycan Receptor Specificity of H2N2 Influenza A Virus Hemagglutinin

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.