Structural basis for the receptor binding specificity of Norwalk virus

Noroviruses are positive-sense, single-stranded RNA viruses that cause acute gastroenteritis. They recognize human histo-blood group antigens as receptors in a strain-specific manner. The structures presented here were analyzed in order to elucidate the structural basis for differences in ligand recognition of noroviruses from different genogroups, the prototypic Norwalk virus (NV; GI-1) and VA387 (GII-4), which recognize the same A antigen but differ in that NV is unable to bind to the B antigen. Two forms of the receptor-binding domain of the norovirus coat protein, the P domain and the P polypeptide, that were previously shown to differ in receptor binding and P-particle formation properties were studied. Comparison of the structures of the NV P domain with and without A trisaccharide and the NV P polypeptide revealed no major ligand-induced changes. The 2.3-A cocrystal structure reveals that the A trisaccharide binds to the NV P domain through interactions with the residues Ser377, Asp327, His329, and Ser380 in a mode distinct from that previously reported for the VA387 P-domain-A-trisaccharide complex. Mutational analyses confirm the importance of these residues in NV P-particle binding to native A antigen. The alpha-GalNAc residue unique to the A trisaccharide is buried deeply in the NV binding pocket, unlike in the structures of A and B trisaccharides bound to VA387 P domain, where the alpha-fucose residue forms the most protein contacts. The A-trisaccharide binding mode seen in the NV P domain complex cannot be sterically accommodated in the VA387 P domain.     

Lewis histo-blood group α1,3/α1,4 fucose residues may both mediate binding to GII.4 noroviruses

Human noroviruses cause recurrent epidemics of gastroenteritis known to be dominated by the clinically important GII.4 genotype which recognizes human Secretor gene-dependent ABH histo-blood group antigens (HBGAs) as attachment factors. There is increasing evidence that GII.4 noroviruses have undergone evolutionary changes to recognize Lewis antigens and non-Secretor saliva. In this study, we have investigated the possibilities of the Lewis α1,3/α1,4 fucoses as mediators of binding of GII.4 noroviruses to Lewis antigens. The study was carried out using molecular dynamics simulations of Lewis type-1 and type-2 chain HBGAs in complex with VA387 P domain dimers in explicit water. Based on the computer simulations, we suggest the possibility of two receptor binding modes for Lewis HBGAs: the "Secretor pose" with the Secretor Fucα1,2 in the binding site and the "Lewis pose" with the Lewis Fucα1,3/α1,4 residues in the binding site. This was further supported by an extensive GlyVicinity analysis of the Protein Data Bank with respect to the occurrence of the Lewis and Secretor poses in complexes of Lewis antigens with lectins and antibodies as well as GII norovirus strains. The Lewis pose can also explain the interactions of GII.4 norovirus strains with Le(x) and SLe(x) structures. Moreover, the present model suggests binding of complex branched polysaccharides, with the Lewis antigens at the nonreducing end, to P domain dimers of GII.4 strains. Our results are relevant for understanding the evolution of norovirus binding specificities and for in silico design of future antiviral therapeutics.     

Evolutionary trace residues in noroviruses: importance in receptor binding, antigenicity, virion assembly, and strain diversity

Noroviruses cause major epidemic gastroenteritis in humans. A large number of strains of these single-stranded RNA viruses have been reported. Due to the absence of infectious clones of noroviruses and the high sequence variability in their capsids, it has not been possible to identify functionally important residues in these capsids. Consequently, norovirus strain diversity is not understood on the basis of capsid functions, and the development of therapeutic compounds has been hampered. To determine functionally important residues in noroviruses, we have analyzed a number of norovirus capsid sequences in the context of the Norwalk virus capsid crystal structure by using the evolutionary trace method. This analysis has identified capsid protein residues that uniquely characterize different norovirus strains and provide new insights into capsid assembly and disassembly pathways and the strain diversity of these viruses. Such residues form specific three-dimensional clusters that may be of functional importance in noroviruses. One of these clusters includes residues known to participate in the proteolytic cleavage of these viruses at high pH. Other clusters are formed in capsid regions known to be important in the binding of antibodies to noroviruses, thereby indicating residues that may be important in the antigenicity of these viruses. The highly variable region of the capsid shows a distinct cluster whose residues may participate in norovirus-receptor interactions.     

Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the polymorphic human histo-blood group antigens

Noroviruses, an important cause of acute gastroenteritis in humans, recognize the histo-blood group antigens (HBGAs) as host susceptible factors in a strain-specific manner. The crystal structures of the HBGA-binding interfaces of two A/B/H-binding noroviruses, the prototype Norwalk virus (GI.1) and a predominant GII.4 strain (VA387), have been elucidated. In this study we determined the crystal structures of the P domain protein of the first Lewis-binding norovirus (VA207, GII.9) that has a distinct binding property from those of Norwalk virus and VA387. Co-crystallization of the VA207 P dimer with Le(y) or sialyl Le(x) tetrasaccharides showed that VA207 interacts with these antigens through a common site found on the VA387 P protein which is highly conserved among most GII noroviruses. However, the HBGA-binding site of VA207 targeted at the Lewis antigens through the α-1, 3 fucose (the Lewis epitope) as major and the β-N-acetyl glucosamine of the precursor as minor interacting sites. This completely differs from the binding mode of VA387 and Norwalk virus that target at the secretor epitopes. Binding pocket of VA207 is formed by seven amino acids, of which five residues build up the core structure that is essential for the basic binding function, while the other two are involved in strain-specificity. Our results elucidate for the first time the genetic and structural basis of strain-specificity by a direct comparison of two genetically related noroviruses in their interaction with different HBGAs. The results provide insight into the complex interaction between the diverse noroviruses and the polymorphic HBGAs and highlight the role of human HBGA as a critical factor in norovirus evolution.     

C-terminal arginine cluster is essential for receptor binding of norovirus capsid protein

Noroviruses are the major viral pathogens of epidemic acute gastroenteritis affecting people worldwide. They have been found to recognize human histo-blood group antigens as receptors. The P domain of norovirus capsid protein was found to be responsible for binding to viral receptors, and the recombinant P protein forms P dimers and P particles in vitro. In this study, we demonstrate that a highly conserved arginine (R) cluster at the C terminus of the P domain is critical for receptor binding and P particle formation of the P proteins. Deletions of the R cluster abolished these functions. Replacement of the R cluster with histidines (another positively charged amino acid) resulted in low efficiency of receptor binding and P particle formation, while replacement with alanines led to loss of both functions completely. The R cluster also contains a highly conserved trypsin digestion site. A treatment of capsid protein or P domain mutants from both genogroup I (Norwalk virus) and genogroup II (VA387) noroviruses with trypsin resulted in a removal of the R cluster and the S domain, leaving a P polypeptide of 31.3 kDa (Norwalk virus) or 34.3 kDa (VA387), similar to the soluble P protein found in vivo. Our findings imply that the proteolytic process could be a necessary step for norovirus replication in the host.     

Norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns

Noroviruses, an important cause of acute gastroenteritis, have been found to recognize human histo-blood group antigens (HBGAs) as receptors. Four strain-specific binding patterns to HBGAs have been described in our previous report. In this study, we have extended the binding patterns to seven based on 14 noroviruses examined. The oligosaccharide-based assays revealed additional epitopes that were not detected by the saliva-based assays. The seven patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H, and Lewis) of human HBGAs: the A/B-binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the norovirus/HBGA interaction. Phylogenetic analyses showed that strains with identical or closely related binding patterns tend to be clustered, but strains in both binding group can be found in both genogroups I and II. Our results suggest that noroviruses have a wide spectrum of host range and that human HBGAs play an important role in norovirus evolution. The high polymorphism of the human HBGA system, the involvement of multiple epitopes, and the typical protein/carbohydrate interaction between norovirus VLPs and HBGAs provide an explanation for the virus-ligand binding diversities.     

Antibody is critical for the clearance of murine norovirus infection

Human noroviruses cause more than 90% of epidemic nonbacterial gastroenteritis. However, the role of B cells and antibody in the immune response to noroviruses is unclear. Previous studies have demonstrated that human norovirus specific antibody levels increase upon infection, but they may not be protective against infection. In this report, we used murine norovirus (MNV), an enteric norovirus, as a model to determine the importance of norovirus specific B cells and immune antibody in clearance of norovirus infection. We show here that mice genetically deficient in B cells failed to clear primary MNV infection as effectively as wild-type mice. In addition, adoptively transferred immune splenocytes derived from B-cell-deficient mice or antibody production-deficient mice were unable to efficiently clear persistent MNV infection in RAG1(-/-) mice. Further, adoptive transfer of either polyclonal anti-MNV serum or neutralizing anti-MNV monoclonal antibodies was sufficient to reduce the level of MNV infection both systemically and in the intestine. Together, these data demonstrate that antibody plays an important role in the clearance of MNV and that immunoglobulin G anti-norovirus antibody can play an important role in clearing mucosal infection.     

The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors

Noroviruses (NVs) are the most important pathogen of epidemic nonbacterial gastroenteritis. The recent finding that NVs recognize human histo-blood group antigens (HBGAs) as receptors provided a new approach to study the pathogenesis of NVs. Using computational and site-directed mutagenesis approaches, our investigators previously identified a plausible binding pocket in the P domain of the NV capsids. In this study, we further characterize the role of the P domain in the interaction with human HBGA receptors using three NV strains representing three binding patterns. Our results show that the isolated P domain, although it did not form virus-like particles (VLPs), formed dimers, and the dimers bound HBGAs with the same patterns as those of the intact viral capsids. In contrast, the S domain, which formed small, thin-layer VLPs, did not bind A, B, or H HBGAs. A chimera containing the S domain of VA387 and the P domain of MOH revealed a binding pattern of the P donor strain (MOH). Deletion experiments revealed that an intact P domain is necessary for receptor binding. The P domain dimers are stable over a broad range of pH (2 to 11) or under strong denaturing conditions. Taken together, our results suggest that the P domain of NV contains essential elements for strain-specific binding to receptors. Further study of the P domain will provide useful information about the virus-receptor interaction. The high yield and easy production of the recombinant P protein in the Escherichia coli expression system will provide a simple approach to this goal.     

Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability

Noroviruses are the dominant cause of outbreaks of gastroenteritis worldwide, and interactions with human histo-blood group antigens (HBGAs) are thought to play a critical role in their entry mechanism. Structures of noroviruses from genogroups GI and GII in complex with HBGAs, however, reveal different modes of interaction. To gain insight into norovirus recognition of HBGAs, we determined crystal structures of norovirus protruding domains from two rarely detected GII genotypes, GII.10 and GII.12, alone and in complex with a panel of HBGAs, and analyzed structure-function implications related to conservation of the HBGA binding pocket. The GII.10- and GII.12-apo structures as well as the previously solved GII.4-apo structure resembled each other more closely than the GI.1-derived structure, and all three GII structures showed similar modes of HBGA recognition. The primary GII norovirus-HBGA interaction involved six hydrogen bonds between a terminal αfucose1-2 of the HBGAs and a dimeric capsid interface, which was composed of elements from two protruding subdomains. Norovirus interactions with other saccharide units of the HBGAs were variable and involved fewer hydrogen bonds. Sequence analysis revealed a site of GII norovirus sequence conservation to reside under the critical αfucose1-2 and to be one of the few patches of conserved residues on the outer virion-capsid surface. The site was smaller than that involved in full HBGA recognition, a consequence of variable recognition of peripheral saccharides. Despite this evasion tactic, the HBGA site of viral vulnerability may provide a viable target for small molecule- and antibody-mediated neutralization of GII norovirus.     

Structural basis for norovirus inhibition and fucose mimicry by citrate

Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 μM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 μM) and H type 2 trisaccharide (390 μM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.     

Murine noroviruses bind glycolipid and glycoprotein attachment receptors in a strain-dependent manner

Human norovirus infections are the most common cause of acute nonbacterial gastroenteritis in humans worldwide, and glycan binding plays an important role in the susceptibility to these infections. However, due to the lack of an efficient cell culture system or small animal model for human noroviruses, little is known about the biological role of glycan binding during infection. Murine noroviruses (MNV) are also enteric viruses that bind to cell surface glycans, but in contrast to their human counterparts, they can be grown in tissue culture and a small animal host. In this study, we determined glycan-binding specificities of the MNV strains MNV-1 and CR3 in vitro, identified molecular determinants of glycan binding, and analyzed infection in vivo. We showed that unlike MNV-1, CR3 binding to murine macrophages was resistant to neuraminidase treatment and glycosphingolipid depletion. Both strains depended on N-linked glycoproteins for binding, while only MNV-1 attachment to macrophages was sensitive to O-linked glycoprotein depletion. In vivo, CR3 showed differences in tissue tropism compared to MNV-1 by replicating in the large intestine. Mapping of a glycan-binding site in the MNV-1 capsid by reverse genetics identified a region topologically similar to the histo-blood group antigen (HBGA)-binding sites of the human norovirus strain VA387. The recombinant virus showed distinct changes in tissue tropism compared to wild-type virus. Taken together, our data demonstrate that MNV strains evolved multiple strategies to bind different glycan receptors on the surface of murine macrophages and that glycan binding contributes to tissue tropism in vivo.     

Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle

Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.     

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m⁷G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.     

Norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle

Recent findings demonstrate that human histo-blood group antigens (HBGAs) serve as receptors for norovirus infection. The recognition of human HBGAs by noroviruses is a typical protein-carbohydrate interaction, in which the protruding domain of the viral capsid protein forms an interface with the oligosaccharide side-chains of the antigens, with a wide diversity among different strains. The human HBGA system is also highly polymorphic and is controlled by multiple gene families with silent alleles. The presence of such diversified molecules on the cell surfaces indicates a possible host defense mechanism against the changing external environment. As mild pathogens that replicate possibly only in the intestinal tract, noroviruses have developed unique strategies to overcome the host defense system. This has been shown by their genetic and structural variations, which explains why norovirus-associated diseases are so common and widespread in every population worldwide.     

Ultrasensitive Norovirus Detection Using DNA Aptasensor Technology

DNA aptamers were developed against murine norovirus (MNV) using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Nine rounds of SELEX led to the discovery of AG3, a promising aptamer with very high affinity for MNV as well as for lab-synthesized capsids of a common human norovirus (HuNoV) outbreak strain (GII.3). Using fluorescence anisotropy, AG3 was found to bind with MNV with affinity in the low picomolar range. The aptamer could cross-react with HuNoV though it was selected against MNV. As compared to a non-specific DNA control sequence, the norovirus-binding affinity of AG3 was about a million-fold higher. In further tests, the aptamer also showed nearly a million-fold higher affinity for the noroviruses than for the feline calicivirus (FCV), a virus similar in size and structure to noroviruses. AG3 was incorporated into a simple electrochemical sensor using a gold nanoparticle-modified screen-printed carbon electrode (GNPs-SPCE). The aptasensor could detect MNV with a limit of detection of approximately 180 virus particles, for possible on-site applications. The lead aptamer candidate and the aptasensor platform show promise for the rapid detection and identification of noroviruses in environmental and clinical samples.     

Norovirus‐binding proteins recovered from activated sludge micro‐organisms with an affinity to a noroviral capsid peptide

Aims: Transmission routes of noroviruses, leading aetiological agents of acute gastroenteritis, are rarely verified when outbreaks occur. Because the destination of norovirus particles being firmly captured by micro‐organisms could be totally different from that of those particles moving freely, micro‐organisms with natural affinity ligands such as virus‐binding proteins would affect the fate of viruses in environment, if such microbial affinity ligands exist. The aim of this study is to identify norovirus‐binding proteins (NoVBPs) that are presumably working as natural ligands for norovirus particles in water environments. Methods and Results: NoVBPs were recovered from activated sludge micro‐organisms by an affinity chromatography technique in which a capsid peptide of norovirus genogroup II (GII) was immobilized. The recovered NoVBPs bind to norovirus‐like particles (NoVLPs) of norovirus GII, and this adsorption was stronger than that to NoVLPs of norovirus genogroup I. The profile of two‐dimensional electrophoresis of NoVBPs showed that the recovered NoVBPs included at least seven spots of protein. The determination of N‐terminal amino acid sequences of these NoVBPs revealed that hydrophobic interactions could contribute to the adsorption between NoVBPs and norovirus particles. Conclusions: NoVBPs conferring a high affinity to norovirus GII were successfully isolated from activated sludge micro‐organisms. Significance and Impact of the Study: NoVBPs could be natural viral ligands and play an important role in the NoV transmission.     

Comparison of human saliva and synthetic histo-blood group antigens usage as ligands in norovirus-like particle binding and blocking assays

Blocking of norovirus-like particle binding to their cellular ligands, histo-blood group antigens with immune sera, is considered a surrogate norovirus neutralization assay. We compared human secretor positive saliva and synthetic biotinylated carbohydrates as a source of histo-blood group antigens in binding and blocking assays. Six norovirus capsid-derived virus-like particles belonging to genogroup I (GI-1-2001 and GI-3-2002) and genogroup II (GII-4-1999, GII-4-2010 New Orleans, GII-4-2012 Sydney and GII-12-1998) noroviruses were produced by a recombinant baculovirus expression system and binding profile to saliva type A, B and O and to synthetic antigens (A trimer, B trimer, H type 1, H type 3, Lewis^a and Lewis^b) was identified. Good correlation between virus-like particle binding to saliva type A and synthetic A trimer (r = 0.66, p < 0.05) and saliva type B and synthetic B trimer (r = 0.75, p < 0.05) was observed. Binding of each norovirus virus-like particle to the selected histo-blood group antigens was blocked by convalescent sera from NoV-infected subjects or type-specific mouse antisera. Our results support the use of either saliva or synthetic antigens in blocking assay to measure the ability of norovirus antisera to block virus-like particle binding to the carbohydrate ligands.     

Cryo-EM Structure of a Novel Calicivirus,Tulane Virus

Tulane virus (TV) is a newly isolated cultivatable calicivirus that infects juvenile rhesus macaques. Here we report a 6.3 Å resolution cryo-electron microscopy structure of the TV virion. The TV virion is about 400 Å in diameter and consists of a T = 3 icosahedral protein capsid enclosing the RNA genome. 180 copies of the major capsid protein VP1 (∼57 KDa) are organized into two types of dimers A/B and C/C and form a thin, smooth shell studded with 90 dimeric protrusions. The overall capsid organization and the capsid protein fold of TV closely resemble that of other caliciviruses, especially of human Norwalk virus, the prototype human norovirus. These close structural similarities support TV as an attractive surrogate for the non-cultivatable human noroviruses. The most distinctive feature of TV is that its C/C dimers are in a highly flexible conformation with significantly reduced interactions between the shell (S) domain and the protruding (P) domain of VP1. A comparative structural analysis indicated that the P domains of TV C/C dimers were much more flexible than those of other caliciviruses. These observations, combined with previous studies on other caliciviruses, led us to hypothesize that the enhanced flexibility of C/C dimer P domains are likely required for efficient calicivirus-host cell interactions and the consequent uncoating and genome release. Residues in the S-P1 hinge between the S and P domain may play a critical role in the flexibility of P domains of C/C dimers.