Site of attachment of encephalomyocarditis virus on human erythrocytes.

Previous studies of the attachment of encephalomyocarditis (EMC) virus to human erythrocytes concluded that the glycophorins, a family of human erythrocyte sialoglycoproteins, act as EMC virus receptors. Evidence is presented that the major glycophorin species, glycophorin A, is the receptor for EMC virus attachment to human erythrocytes. Comparison of the structures of glycophorins A and B and sialoglycopeptides released by chymotrypsin and trypsin treatment of erythrocytes confirmed our previous suggestion (A. T. H. Burness and I. U. Pardoe, J. Gen. Virol. 64:1137-1148, 1983) that attachment of EMC virus to glycophorin A involves the region containing amino acids 35 to approximately 70 (numbered from the NH2 terminus), four of which (amino acids 37, 44, 47, and 50) are glycosylated. In addition, we provide evidence that the segment containing amino acids 35 to 39 with an oligosaccharide side chain on threonine-37 is particularly important for EMC virus attachment.     

Conformational alterations within the glycocalyx of erythrocyte membranes studied by spin labelling

The structure of the glycocalyx of the membrane of human erythrocytes and spectrin-depleted vesicles was studied under various conditions by two spin-labelling approaches: covalently labelling sialic acid residues of the glycocalyx and incorporation of a charged hydrophobic spin probe, CAT 16, being sensitive to alterations on the membrane surface into the lipid phase. Although cell electrophoretic measurements which were performed, additionally, indicated an erection of the glycocalyx upon decreasing the ionic strength of the suspension medium a more restricted mobility of spin-labelled sialic acid residues was found, in this case probably due to electrostatic interactions. The enhanced mobility of the spin probe CAT 16 at low ionic strength as well as in the case of neuraminidase-treated cells could be caused by reduced steric and electrostatic interaction with glycoproteins and glycolipids. La3+ adsorption and virus attachment on the human erythrocyte membrane were accompanied with a reduced mobility of sugar headgroups of the surface coat. No indication of cluster formation or lateral segregation of glycophorin molecules was found upon virus binding. After denaturation of the spectrin cytoskeleton of intact erythrocytes, increased mobility of spin-labelled sialic acid residues was observed.     

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.     

Sialic acid functions in enterovirus 70 binding and infection

The interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decay-accelerating factor (DAF/CD55) is an attachment receptor for enterovirus 70 (EV70), a member of the Picornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.     

Contact residues and predicted structure of the reovirus type 3-receptor interaction.

Sequence similarity between the reovirus type 3 hemagglutinin (HA3) and a anti-idiotypic monoclonal antibody (87.92.6) has been shown to define the site of interaction with a neutralizing (idiotypic) monoclonal antibody (9B.G5) and the cellular receptor for the virus. A synthetic peptide (VL peptide) derived from the anti-idiotypic sequence inhibits viral binding to the receptor. In this study, variants of the VL peptide were utilized to probe specific amino acid residues involved in binding the neutralizing antibody and the receptor. These studies indicate that the--OH groups of several residues are involved in contacting the reovirus type 3 receptor, including Tyr49, Ser50, Ser52, and Thr53 in the anti-idiotypic sequence, corresponding to Tyr326, Ser327, Ser329, and Ser325 in HA3, respectively. In contrast, only Ser50 of the anti-idiotypic sequence, corresponding to Ser327 of HA3, significantly altered neutralizing antibody binding. Additional studies implicate sialic acid as a potential reovirus type 3 receptor on some cells. This includes inhibition of binding of reovirus type 3 and 87.92.6 to L cells by heavily sialylated glycoproteins. Sialic acid was therefore utilized as a candidate receptor to analyze potential interaction schemes with HA3/87.92.6. Sequence similarity to other immunoglobulin structures with similar sequences allowed modeling of the three-dimensional structure of these epitopes. These structures, in combination with peptide studies, allow the development of a model of the interaction of these epitopes with sialic acid, which serves as a reovirus type 3 receptor. These models reveal that similar amino acid residues and side-chain geometries may be utilized by the reovirus type 3 and influenza hemagglutinins in their interactions with cell-surface receptors.     

Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation

The alpha-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228-Leu248) to intensive random mutagenesis and screened multiply substituted alleles for receptor function. The 91 partially functional mutant alleles that were recovered contained 96 unique amino acid substitutions. Every position in this region can be replaced with at least two other types of amino acids without a significant effect on function. The tolerance for nonconservative substitutions indicates that activation of the G protein by ligand-bound receptors involves multiple intramolecular interactions that do not strongly depend on particular sequence elements. Many of the functional mutant alleles exhibit greater than normal levels of signaling, consistent with an inhibitory role for the third intracellular loop. Removal of increasing numbers of positively charged residues from the loop by site-directed mutagenesis causes a progressive loss of signaling function, indicating that the overall net charge of the loop is important for receptor function. Introduction of negatively charged residues also leads to a reduced level of signaling. The defects in signaling caused by substitution of charged amino acids are not caused by changes in the abundance of receptors at the cell surface.     

Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening

Many serotype 3 reoviruses bind to two different host cell molecules, sialic acid and an unidentified protein, using discrete receptor-binding domains in viral attachment protein, final sigma1. To determine mechanisms by which these receptor-binding events cooperate to mediate cell attachment, we generated isogenic reovirus strains that differ in the capacity to bind sialic acid. Strain SA+, but not SA-, bound specifically to sialic acid on a biosensor chip with nanomolar avidity. SA+ displayed 5-fold higher avidity for HeLa cells when compared with SA-, although both strains recognized the same proteinaceous receptor. Increased avidity of SA+ binding was mediated by increased k(on). Neuraminidase treatment to remove cell-surface sialic acid decreased the k(on) of SA+ to that of SA-. Increased k(on) of SA+ enhanced an infectious attachment process, since SA+ was 50-100-fold more efficient than SA- at infecting HeLa cells in a kinetic fluorescent focus assay. Sialic acid binding was operant early during SA+ attachment, since the capacity of soluble sialyllactose to inhibit infection decreased rapidly during the first 20 min of adsorption. These results indicate that reovirus binding to sialic acid enhances virus infection through adhesion of virus to the cell surface where access to a proteinaceous receptor is thermodynamically favored.     

Conserved negatively charged residues are not required for ATP action at P2X(1) receptors.

The role of conserved negatively charged aspartic (D) and glutamic (E) acid residues within the ectodomain of the human P2X(1) receptor were examined by alanine substitution mutagenesis. Effects on ATP potency and cell surface localisation were assessed in Xenopus oocytes using the two electrode voltage clamp technique and cell surface biotinylation. Of the eleven residues tested no major shifts in ATP potency were observed with EC(50) values for ATP ranging from 0.8 to 4.3 microM (compared to 1 microM ATP for wild-type P2X(1) receptors). Peak current amplitudes for mutants D86A and D264A where reduced by approximately 90% due to a corresponding reduction in both total protein and cell surface expression. These results demonstrate that individual conserved negatively charged amino acids are not essential for ATP recognition by the human P2X(1) receptor and coordinated binding of the positive charge on magnesium complexed ATP by negatively charged amino acids is not required.     

Neutralization map of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus: domains recognized by monoclonal antibodies that prevent receptor recognition.

Monoclonal antibodies (MAbs) to the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus delineate seven overlapping antigenic sites which form a continuum on the surface of the molecule. Antibodies to five of these sites neutralize viral infectivity principally by preventing attachment of the virion to cellular receptors. Through the identification of single amino acid substitutions in variants which escape neutralization by MAbs to these five antigenic sites, a neutralization map of HN was constructed, identifying several residues that contribute to the epitopes recognized by MAbs which block the attachment function of the molecule. These epitopes are defined, at least in part, by three domains on HN: residues 193 to 201; 345 to 353 (which include the only linear epitope we have identified in HN); and a C-terminal domain composed of residues 494, 513 to 521, and 569. To identify HN residues directly involved in receptor recognition, each of the variants was tested for its ability to agglutinate periodate-modified chicken erythrocytes. One variant with a single amino acid substitution at residue 193 was 2.5- to 3-fold more resistant to periodate treatment of erythrocytes than the wild-type virus, suggesting that this residue influences the binding of virus to a sialic acid-containing receptor(s) on the cell surface.     

Trypanosoma cruzi trans-sialidase and cell invasion

Trypanosoma cruzi does not synthesize sialic acid but does contain a trans-sialidase, an enzyme capable of transferring sialic acid between host glycoconjugates and the parasite. Sialic acids are negatively charged carbohydrates attached to the terminal non-reducing end of glycoproteins and glycolipids, and their presence can dramatically influence many cell-surface recognition processes. Since sialic acids have been implicated in several ligand-receptor interactions, including the interaction of pathogenic viruses, bacteria and protozoans with their hosts, the expression of trans-sialidase and the acquisition of sialic acid by T. cruzi may be relevant to the interaction of the parasite with the host, and consequently may influence the pathobiology of Chagas disease. In this review, Sergio Schenkman and Daniel Eichinger discuss recent data about the structure and function of T. cruzi trans-sialidase.     

Structural requirements for glycosyl-phosphatidylinositol-anchor attachment in the cellular receptor for urokinase plasminogen activator.

The urokinase-plasminogen-activator receptor (u-PAR) is a glycosyl-phosphatidylinositol(glycosyl-PtdIns)-anchored membrane protein. Using site-directed mutagenesis, we have studied features in the u-PAR sequence important for successful glycosyl-PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282-Gly283-Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl-PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl-PtdIns-attachment site. In the last eight COOH-terminal amino acids encoded in u-PAR cDNA, deletion of this sequence (residues 306-313) completely prevented glycosyl-PtdIns attachment. However, the remaining COOH-terminal region proved still to possess a potential glycosyl-PtdIns signal activity; it could be converted to a new functional glycosyl-PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u-PAR to a glycosyl-PtdIns-anchored protein, indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial acquisition of the glycosyl-PtdIns-anchoring ability. These findings show that charged amino acids placed in the COOH-terminus interfere negatively with glycosyl-PtdIns-anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.     

Role of sialic acid-containing molecules in paramyxovirus entry into the host cell: A minireview

Sialic acid-containing compounds play a key role in the initial steps of the paramyxovirus life cycle. As enveloped viruses, their entry into the host cell consists of two main events: binding to the host cell and membrane fusion. Virus adsorption occurs at the surface of the host cell with the recognition of specific receptor molecules located at the cell membrane by specific viral attachment proteins. The viral attachment protein present in some paramyxoviruses (Respirovirus, Rubulavirus and Avulavirus) is the HN glycoprotein, which binds to cellular sialic acid-containing molecules and exhibits sialidase and fusion promotion activities. Gangliosides of the gangliotetraose series bearing the sialic acid N-acetylneuraminic (Neu5Ac) on the terminal galactose attached in α2-3 linkage, such as GD1a, GT1b, and GQ1b, and neolacto-series gangliosides are the major receptors for Sendai virus. Much less is known about the receptors for other paramyxoviruses than for Sendai virus. Human parainfluenza viruses 1 and 3 preferentially recognize oligosaccharides containing N-acetyllactosaminoglycan branches with terminal Neu5Acα2-3Gal. In the case of Newcastle disease virus, has been reported the absence of a specific pattern of the gangliosides that interact with the virus. Additionally, several works have described the use of sialylated glycoproteins as paramyxovirus receptors. Accordingly, the design of specific sialic acid analogs to inhibit the sialidase and/or receptor binding activity of viral attachment proteins is an important antiviral strategy. In spite of all these data, the exact nature of paramyxovirus receptors, apart from their sialylated nature, and the mechanism(s) of viral attachment to the cell surface are poorly understood. The authors would like to dedicate this review to Prof. José A. Cabezas, recently retired who, as well being our mentor and colleague, introduced us into the fascinating field of sialic acid-containing glycoconjugates and viral sialidases at a time when just a very small number of scientists were paying attention to this important field of research. Also, he has been for us a continuous source of inspiration and friendship to us. The ganglioside nomenclature of Svennerholm [1] is used.     

N-acetyl-9-O-acetylneuraminic acid, the receptor determinant for influenza C virus, is a differentiation marker on chicken erythrocytes

Erythrocytes from chicken of different age were analysed for their agglutinability by influenza C virus, which has been shown recently to use N-acetyl-9-O-acetylneuraminic acid as a high-affinity receptor determinant for the attachment to cells. Only with birds not younger than six days complete agglutination of the erythrocytes was observed. The hemagglutination titer which was initially low reached its maximum value at the age of about 20 days. Sialic acid was isolated from erythrocytes, purified and analysed by colorimetry, thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. The sialic acid content of erythrocytes from one-day old and adult chicken was 21 micrograms and 18 micrograms sialic acid/ml packed erythrocytes, respectively. While N-acetylneuraminic acid was the major type of sialic acid on erythrocytes from both one-day old and adult chicken, N-acetyl-9-O-acetylneuraminic acid was only detected on red blood cells from adult animals accounting for 30-40% of total sialic acid. These results indicate that N-acetyl-9-O-acetylneuraminic acid, in addition to serving as a receptor determinant for influenza C virus, represents a developmental marker on chicken erythrocytes.     

Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses

Virus attachment to host cells is mediated by dedicated virion proteins, which specifically recognize one or, at most, a limited number of cell surface molecules. Receptor binding often involves protein-protein interactions, but carbohydrates may serve as receptor determinants as well. In fact, many different viruses use members of the sialic acid family either as their main receptor or as an initial attachment factor. Sialic acids (Sias) are 9-carbon negatively-charged monosaccharides commonly occurring as terminal residues of glycoconjugates. They come in a large variety and are differentially expressed in cells and tissues. By targeting specific Sia subtypes, viruses achieve host cell selectivity, but only to a certain extent. The Sia of choice might still be abundantly present on non-cell associated molecules, on non-target cells (including cells already infected) and even on virus particles themselves. This poses a hazard, as high-affinity virion binding to any of such “false' receptors would result in loss of infectivity. Some enveloped RNA viruses deal with this problem by encoding virion-associated receptor-destroying enzymes (RDEs). These enzymes make the attachment to Sia reversible, thus providing the virus with an escape ticket. RDEs occur in two types: neuraminidases and sialate-O-acetylesterases. The latter, originally discovered in influenza C virus, are also found in certain nidoviruses, namely in group 2 coronaviruses and in toroviruses, as well as in infectious salmon anemia virus, an orthomyxovirus of teleosts. Here, the structure, function and evolution of viral sialate-O-acetylesterases is reviewed with main focus on the hemagglutinin-esterases of nidoviruses.     

Amino acid substitutions in VP2 residues contacting sialic acid in low-neurovirulence BeAn virus dramatically reduce viral binding and spread of infection

Theiler's murine encephalomyelitis viruses (TMEV) consist of two groups, the high- and low-neurovirulence groups, based on lethality in intracerebrally inoculated mice. Low-neurovirulence TMEV result in a persistent central nervous system infection in mice, leading to an inflammatory demyelinating pathology and disease. Low- but not high-neurovirulence strains use sialic acid as an attachment factor. The recent resolution of the crystal structure of the low-neurovirulence DA virus in complex with the sialic acid mimic sialyllactose demonstrated that four capsid residues make contact with sialic acid through noncovalent hydrogen bonds. To systematically test the importance of these sialic acid-binding residues in viral entry and infection, we mutated three VP2 puff B amino acids proposed to make contact with sialic acid and analyzed the consequences of each amino acid substitution on viral entry and spread. The fourth residue is in the VP3-VP1 cleavage dipeptide and could not be mutated. Our data suggest that residues Q2161 and G2174 are directly involved in BeAn virus attachment to sialic acid and that substitutions of these two residues result in the loss of or reduced viral binding and hemagglutination and in the inability to spread among BHK-21 cells. In addition, a gain of function-revertant virus was recovered with the Q2161A mutation after prolonged passage in cells.     

Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity

Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.     

Enhanced sialic acid-dependent endocytosis explains the increased efficiency of infection of airway epithelia by a novel adeno-associated virus

We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.     

Sialic acid metabolism and sialyltransferases: natural functions and applications

Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.