The effect of cellular receptor diffusion on receptor-mediated viral binding using Brownian adhesive dynamics (BRAD) simulations

Brownian adhesive dynamics (BRAD) is a new method for simulating the attachment of viruses to cell surfaces. In BRAD, the motion of the virus is subject to stochastic bond formation and breakage, and thermal motion owing to collisions from the solvent. In the model, the virus is approximated as a rigid sphere and the cell surface is approximated as a rigid plane coated with receptors. In this article, we extend BRAD to allow for the mobility of receptors in the plane of the membrane, both before and after they are ligated by viral attachment proteins. Allowing the proteins to move within the membrane produced several differences in behavior from when the receptors are immobilized. First, the mean steady-state bond number is unaffected by changes in cellular receptor density because proteins are now free to diffuse into the contact area, and the extent of binding is dictated by the availability of viral attachment proteins. Second, the time required to reach steady-state binding increases as both the cellular receptor number decreases and the receptor mobility decreases. This is because receptor diffusion is a slower process than the binding kinetics of the proteins. Decreasing the rate of protein binding was found to decrease the fraction of viruses bound to steady state, but not the extent of binding for those viruses that were bound. Increasing the binding rate increased the fraction of viruses bound, until no further viruses could bind. Alterations in receptor binding kinetics had no discernable effect on the mean steady-state bond number between virus and cell, because interactions were of sufficiently high affinity that all available receptor-viral attachment proteins were destined to bind at steady state.     

General analysis of receptor-mediated viral attachment to cell surfaces.

Viruses are multivalent particles that attach to cells through one or more bonds between viral attachment proteins (VAP) and specific cellular receptors. Three modes of virus binding are presented that can explain the diversity in binding data observed among viruses. They are based on multivalency of attachment and spatial versus receptor saturation effects which are easily distinguished based upon simple criteria. Mode 1 involves only monovalent virus/receptor binding. Modes 2 and 3 involve multivalent bonds between the virus and cell; however, in mode 3 space on the cell surface becomes saturated before receptors. A model is developed for viral attachment that accounts for nonspecific binding, receptor/virus interactions, and spatial saturation effects. The model can describe each mode in different limits and can be applied to virus binding data to extract key physical information such as receptor number and affinity. These values are used to postulate the type of VAP/receptor interaction involved and to predict binding at different parameter values. For the mode 2 binding of Adenovirus 2, the model predicts a receptor number of 4-15 x 10(3) on HeLa cells and an affinity of 2-6 x 10(7) M-1 which closely approximate experimental estimates. For the binding of three, broad-host-range, enveloped viruses, Semliki Forest virus, Vesicular Stomatitis virus, and the baculovirus, Autographa californica nuclear polyhedrosis virus, the model predicts receptor numbers of 10(5) or greater and affinities in the range of 10(4) to 10(5) M-1. These values are indicative of a VAP/oligosaccharide interaction which has been documented for a number of other viruses. Experimental evidence is presented that is the first to demonstrate that baculovirus binding is mediated by a cell surface receptor.     

The multiple facets of HIV attachment to dendritic cell lectins

Entry of enveloped viruses into host cells depends on the interactions of viral surface proteins with cell surface receptors. Many enveloped viruses maximize the efficiency of receptor engagement by first binding to attachment‐promoting factors, which concentrate virions on target cells and thus increase the likelihood of subsequent receptor engagement. Cellular lectins can recognize glycans on viral surface proteins and mediate viral uptake into immune cells for subsequent antigen presentation. Paradoxically, many viral and non‐viral pathogens target lectins to attach to immune cells and to subvert cellular functions to promote their spread. Thus, it has been proposed that attachment of HIV to the dendritic cell lectin DC‐SIGN enables the virus to hijack cellular transport processes to ensure its transmission to adjacent T cells. However, recent studies show that the consequences of viral capture by immune cell lectins can be diverse, and can entail negative and positive regulation of viral spread. Here, we will describe key concepts proposed for the role of lectins in HIV attachment to host cells, and we will discuss recent findings in this rapidly evolving area of research.     

Characterization of a common high-affinity receptor for reovirus serotypes 1 and 3 on endothelial cells.

During viremia, viruses may be cleared from the bloodstream and taken up by specific organs. The uptake of virus from the bloodstream is dependent on the association of viral particles with endothelial cells that line the luminal surfaces of large and small blood vessels. To understand the nature of this interaction, we have studied the binding of reovirus serotypes 1 and 3 to these cells in vitro. Both serotypes of reovirus productively infected endothelial cells. By using [35S]methionine-biolabeled reovirus as a tracer ligand, we found that both viruses rapidly bind to endothelial cells and that equilibrium is reached after 4 h. The binding of the radiolabeled viruses was saturable and mediated by a homogeneous population of cellular receptors with very high affinity (Kd = 0.5 nM) for the virus ligands. Both serotypes bind to the same receptor, since the attachment of each radiolabeled serotype is inhibited by both the homologous and heterologous unlabeled virus. Exposure of labeled virus to monoclonal antibodies directed against the viral hemagglutinin (sigma 1 protein) inhibited binding, demonstrating that the attachment of reovirus to endothelial cells is mediated by the hemagglutinin for both serotypes. By using a novel ligand-blotting assay, the binding of both viruses to a 54,000-dalton protein could be demonstrated. The binding of each radiolabeled serotype to this protein was inhibited by the homologous and heterologous unlabeled serotype. By using cell fractionation after homogenization, we demonstrated that this 54-kilodalton protein is a membrane protein, in agreement with its proposed role as a cell surface receptor for reovirus serotypes 1 and 3.     

The canyon hypothesis. Hiding the host cell receptor attachment site on a viral surface from immune surveillance

The three-dimensional structure of human rhinovirus 14 has a deep surface depression or "canyon" encircling each of the twelve 5-fold vertices. The canyon's surface is inaccessible to the broad antigen binding region of antibodies, permitting conservation of residues that might be required for host cell receptor recognition without danger of attack by the host's immune system. In contrast, the exposed surface features, where neutralizing antibodies are known to bind, change rapidly under pressure from the host's immune system. It was, therefore, hypothesized that this depression was the site of receptor attachment. Similar, but smaller, depressions had been observed previously on both the hemagglutinin and neuraminidase spikes of influenza virus. These have also been shown to be the site of host cell interaction. Although support for the canyon hypothesis was only circumstantial in the first place, there are now extensive confirmatory data. These include site-specific mutations of residues in the canyon and conformational changes induced in the canyon by the binding of small organic molecules, all of which alter receptor attachment. The strategy used in human rhinovirus 14 to protect the viral receptor attachment site from immune surveillance may be utilized not only in other picornaviruses but also in many other types of viruses including human immunodeficiency virus.     

Mutation of predicted virion pit residues alters binding of Theiler's murine encephalomyelitis virus to BHK-21 cells

Theiler's murine encephalomyelitis virus (TMEV), a natural pathogen of mice, is a member of the genus Cardiovirus in the family Picornaviridae. Structural studies indicate that the cardiovirus pit, a deep depression on the surface of the virion, is involved in receptor attachment; however, this notion has never been systematically tested. Therefore, we used BeAn virus, a less virulent TMEV, to study the effect of site-specific mutation of selected pit amino acids on viral binding as well as other replicative functions of the virus. Four amino acids within the pit, V1091, P1153, A1225 and P3179, were selected for mutagenesis to evaluate their role in receptor attachment. Three amino acid replacements were made at each site, the first a conservative replacement, followed by progressively more radical amino acid changes in order to detect variable effects at each site. A total of seven viable mutant viruses were recovered and characterized for their binding properties to BHK-21 cells, capsid stability at 40 degrees C, viral RNA replication, single- and multistep growth kinetics, and virus translation. Our data implicate three of these residues in TMEV-cell receptor attachment.     

A fifteen-amino-acid TVB peptide serves as a minimal soluble receptor for subgroup B avian leukosis and sarcoma viruses

The TVB receptor for subgroup B, D, and E avian sarcoma and leukosis viruses (ASLVs) is a tumor necrosis factor receptor-related protein that is most closely related to the TRAIL receptors. Here we show that the major subgroup B viral interaction determinants of TVB are contained within a linear 15-amino-acid peptide derived from the N-terminal region of the receptor. Moreover, this peptide was sufficient not only for binding to ASLV-B but also for activating viral entry into mammalian cells that lacked the cognate viral receptor. Peptide-dependent viral entry was blocked in the presence of bafilomycin A1, indicating that virions can be trafficked to an acidic endosomal fusion compartment without the need for physical attachment of the viral receptor to a cellular membrane.     

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.     

Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.     

Structural and mechanistic studies of measles virus illuminate paramyxovirus entry

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.     

Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.     

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.     

Residue Y161 of influenza virus hemagglutinin is involved in viral recognition of sialylated complexes from different hosts

Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to N-acetylneuraminic acid α3 (NeuAcα3) sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species-specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied, and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved HA surface-located amino acid residues in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell-type-dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharide binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to N-glycolylneuraminic acid (NeuGc). Rescued mutant Y161A viruses demonstrated a 5- to 10-fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism.     

X-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein

Although many viral receptors have been identified, the ways in which they interact with their cognate viruses are not understood at the molecular level. We have determined the X-ray structure of a complex between calcium-containing modules of the very low-density lipoprotein receptor and the minor group human rhinovirus HRV2. The receptor binds close to the icosahedral five-fold vertex, with only one module per virus protomer. The binding face of this module is defined by acidic calcium-chelating residues and, in particular, by an exposed tryptophan that is highly conserved. The attachment site on the virus involves only residues from VP1, particularly a lysine strictly conserved in all minor group HRVs. The disposition of the attached ligand-binding repeats around the five-fold axis, together with the proximity of the N- and C-terminal ends of adjacent modules, suggests that more than one repeat in a single receptor molecule might attach simultaneously.     

Host-related immunomodulators encoded by poxviruses and herpesviruses

In the past year, important advances have been made in the area of host-related immunomodulatory genes encoded by the larger DNA viruses, particularly for the poxviruses and herpesviruses. Not only has the repertoire of viral immunomodulator homologs expanded as a result of sequencing the complete genome of another six, large DNA viruses, but also new concepts of how they work have been proposed and in some cases supported by in vivo evidence. Recent developments have been made in understanding a spectrum of host-related viral modulators, including complement control proteins, TNF-receptor homologs, IL-18 binding proteins, viral interleukins (vIL-6 and vIL-10), chemokine mimics and chemokine receptor homologs.     

The role of cellular adhesion molecules in virus attachment and entry

As obligate intracellular parasites, viruses must traverse the host-cell plasma membrane to initiate infection. This presents a formidable barrier, which they have evolved diverse strategies to overcome. Common to all entry pathways, however, is a mechanism of specific attachment to cell-surface macromolecules or ‘receptors’. Receptor usage frequently defines viral tropism, and consequently, the evolutionary changes in receptor specificity can lead to emergence of new strains exhibiting altered pathogenicity or host range. Several classes of molecules are exploited as receptors by diverse groups of viruses, including, for example, sialic acid moieties and integrins. In particular, many cell-adhesion molecules that belong to the immunoglobulin-like superfamily of proteins (IgSF CAMs) have been identified as viral receptors. Structural analysis of the interactions between viruses and IgSF CAM receptors has not shown binding to specific features, implying that the Ig-like fold may not be key. Both proteinaceous and enveloped viruses exploit these proteins, however, suggesting convergent evolution of this trait. Their use is surprising given the usually occluded position of CAMs on the cell surface, such as at tight junctions. Nonetheless, the reason for their widespread involvement in virus entry most probably originates in their functional rather than structural characteristics.     

Virus–Receptor Interactions: The Key to Cellular Invasion

Virus–receptor interactions play a key regulatory role in viral host range, tissue tropism, and viral pathogenesis. Viruses utilize elegant strategies to attach to one or multiple receptors, overcome the plasma membrane barrier, enter, and access the necessary host cell machinery. The viral attachment protein can be viewed as the “key” that unlocks host cells by interacting with the “lock”—the receptor—on the cell surface, and these lock-and-key interactions are critical for viruses to successfully invade host cells. Many common themes have emerged in virus–receptor utilization within and across virus families demonstrating that viruses often target particular classes of molecules in order to mediate these events. Common viral receptors include sialylated glycans, cell adhesion molecules such as immunoglobulin superfamily members and integrins, and phosphatidylserine receptors. The redundancy in receptor usage suggests that viruses target particular receptors or “common locks” to take advantage of their cellular function and also suggests evolutionary conservation. Due to the importance of initial virus interactions with host cells in viral pathogenesis and the redundancy in viral receptor usage, exploitation of these strategies would be an attractive target for new antiviral therapeutics.     

Attachment of VLDL receptors to an icosahedral virus along the 5-fold symmetry axis: multiple binding modes evidenced by fluorescence correlation spectroscopy

Human rhinoviruses (HRVs) are composed of 60 identical subunits, each comprising one copy of the viral capsid proteins VP1, 2, 3, and 4. Consequently, 60 symmetry-related epitopes are available for binding of antibodies or receptors. The minor receptor group of HRVs uses members of the low-density lipoprotein receptor family for cell entry. The ligand binding domains of these receptors are composed of various numbers of ligand binding repeats, and several of these modules within a single molecule are believed to attach simultaneously to the star-shaped dome at the 5-fold symmetry axis of the virus. Using fluorescence correlation spectroscopy (FCS), we have now determined the equilibrium binding constants and the mode of attachment of recombinant concatemers of ligand binding module 3 of the human very-low-density lipoprotein receptor to HRV2. We demonstrate that the avidity of the interaction drastically increases with the number of concatenated modules. For the trimer, the binding isotherm was biphasic, indicating that attachment of two and of three modules within the same molecule was resolved. The receptor consisting of seven repeats was found to bind most strongly, but a complete binding isotherm could not be established due to cross-linking of virions. The values of the dissociation constants were about 1 order of magnitude higher than those previously determined by using surface plasmon resonance techniques reflecting the different presentation of the binding partners. As compared to the concatemers, the natural receptors are composed of similar but not identical repeats; thus, cooperativity and different specificity of the ligand-binding modules allow for recognition of many ligands and viral serotypes. Due to the low concentrations and amounts of sample required, FCS is ideally suited for the determination of receptor binding parameters of viruses difficult to produce in high quantities and/or concentrations.     

Mouse cells expressing human intercellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21.

Competitive viral binding assays have revealed previously that coxsackievirus A21 (CAV21) and human rhinovirus 14 (HRV14) share a common cell surface receptor. More recently, intercellular adhesion molecule-1 (ICAM-1) has been identified as the cellular receptor for HRV-14. Also, anti-ICAM-1 monoclonal antibodies (MAbs) blocked infection by HRV14, CAV13, CAV18, and CAV21, suggesting that these viruses share this receptor; however, this has never been established by more direct methods. In this study we show conclusively that CAV21 binds to ICAM-1 and that MAbs directed against the N-terminal domain of the molecule inhibit this attachment. Furthermore, we show that the specific interaction between ICAM-1 and 160S CAV21 virions induces formation of 135S A particles. Finally, we show transfection of normally nonsusceptible mouse L cells with human ICAM-1 cDNA renders them susceptible to infection by CAV21.     

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.