Human Norovirus Receptors

Chapter 3.3

Human Norovirus Receptors

J. Le Pendu*

G.E. Rydell**

W. Nasir

G. Larson
*    Inserm, CNRS, Nantes University, IRS UN, Nantes, France
**    Department of Infectious Diseases, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
    Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden


Due to the lack of cell culture methods, studies of human norovirus receptors have been limited to virus challenge and outbreak studies and in vitro binding studies of virus-like particles (VLPs). Norovirus VLPs recognize glycans from the ABO(H) and Lewis histo-blood group family on glycoproteins and glycosphingolipids in a strain-dependent manner. Host genetic studies have shown that for the clinically dominating strains the binding pattern correlates with susceptibility to infection. Particularly, the so-called nonsecretors, characterized by their lack of expression of ABO(H) structures in the gastrointestinal tract, have been identified as resistant to infections by many strains. In support of a receptor status, norovirus VLPs have been shown to bind to intestinal epithelium expressing ABO(H) epitopes, to intestinal glycosphingolipids carrying ABO(H) epitopes and to induce membrane invaginations, resembling endocytosis intermediates, on model membranes carrying such glycosphingolipids. However, in addition to the ABO(H) structures, the VLPs also recognizes other glycans that could play an important role in virus entry into the host cells.








1. Introduction

Human noroviruses (NoVs) are a leading cause of viral gastroenteritis, infecting all age groups worldwide and as such represent a major public health problem. Despite being mainly transmitted through person-to-person contact these viruses are the principal cause of toxic food-borne viral infections in Europe and in the United States (Ahmed et al., 2014Hall et al., 2012Patel et al., 2009). Although they generally cause a relatively mild disease of short duration, NoVs can be responsible for severe dehydration that may lead to hospitalization and even death of at-risk patients, notably young children, elderly people in poor health and immunocompromised patients (Bok and Green, 2012Gustavsson et al., 2011). Thus, these small nonenveloped RNA viruses represent the main cause of acute gastroenteritis leading to hospitalization in adults and the second leading cause after rotavirus in young children in the United States (Hall, 2012). In countries with established universal rotavirus vaccination programs, NoVs are the most frequent cause of acute gastroenteritis also in children (Payne et al., 2013Koo et al., 2013Hemming et al., 2013Bucardo et al., 2014). NoV present with a very large genetic diversity. By comparing sequences that code for the polymerase and the capsid protein of a large number of human and animal NoV strains, they have been classified into distinct groups (genogroups GI–GVI). GI and GII NoVs include strains that are the most frequently implicated in human infections. GIII exclusively comprises bovine strains. The GIV genogroup is rarely found in humans and mainly includes strains isolated in lions and dogs. GV and GVI include murine and canine strains, respectively (Mesquita et al., 2010). Finally, the existence of a seventh genogroup has been proposed after the discovery of new canine strains (Vinjé, 2015). In each genogroup, the analysis of complete ORF2 sequences that encode the capsid protein has revealed huge genetic diversity, making it possible to distinguish between eight genotypes in GI (GI.1–GI.8) and 21 genotypes in GII (GII.1–GII.21). GII NoVs are primarily found in humans. The three exceptions are GII.11, GII.18, and GII.19, which are exclusively found in pigs (Hall et al., 2011Zheng et al., 2006). From an epidemiological point of view, GII NoVs are currently predominant. Among these, the GII.4 genotype alone is responsible for over 70% of all cases of NoV gastroenteritis (Glass et al., 2009Hall et al., 2011). (See also Chapter 3.5).

The first step of a viral infection is characterized by attachment onto a susceptible cell through surface components that will contribute to virus entry. Those cell surface components involved in the process of binding and entry function as viral receptors. Yet, in many instances, binding to a single attachment or adhesion factor is not sufficient for infection since additional molecules, or coreceptors are required for entry to take place (Verdaguer et al., 2014). In the case of human NoVs, recognition and binding to several glycans has been demonstrated. However, their exact role in the infection process remains ill-defined since until very recently it has not been possible to cultivate human NoVs, hampering studies of the entry process.

Following the discovery that Rabbit Hemorrhagic Disease Virus (RHDV), another member of the Caliciviridae family, could attach to glycans of the histo-blood group family (HBGAs) (Ruvoën-Clouet et al., 2000), it was observed that human NoVs recognize similar carbohydrates (Marionneau et al., 2002). It was then suggested that human NoVs could additionally bind to heparan sulfate (Tamura et al., 2004) and sialylated glycans such as sialyl Lex (SLex) and sialyl-type 2 precursor (Rydell et al., 2009b). Very recently, specific NoV recognition of gangliosides, that is, glycosphingolipids carrying one (or more) sialic acids, was demonstrated in addition to that of HBGAs (Han et al., 2014), an intriguing observation reminiscent of the recognition of HBGAs and gangliosides by various strains of rotavirus (Hu et al., 2012Huang et al., 2012Martinez et al., 2013).

2. Human norovirus binds to histo-blood group antigens

HBGAs, which include the ABH, Lewis, I/i, and P antigens, are complex sugars typically present on the erythrocytes of humans and some of the great apes (Storry and Olsson, 2004). However, HBGAs are also found on the surface of epithelial cells of different tissues in a wide range of vertebrate species and can be secreted in free or complex forms in biological fluids such as saliva and milk (Marionneau et al., 2001). Since most human NoVs recognize ABH and/or Lewis antigens we will concentrate on these carbohydrate motifs. Their synthesis is under the control of several genes that code for glycosyltransferases and display common genetic polymorphisms (Lowe, 1993) (Fig. 3.3.1).


Figure 3.3.1 Biosynthetic pathways of HBGAs.
The precursors from type-1 and type-2 chains glycans are considered. The names of antigens (blue boxes), genetic loci of glycosyltransferases (italic) and the glycosidic linkages (solid lines) are separated by forward slash (/) to specify the pathways for either chain type. The FUT2 and the FUT3 genetic loci driving the biosynthesis of the secretor (Se) and the Lewis (Le) antigens, respectively, are also highlighted (bold).

The α1,2-fucosyltransferase encoded by the FUT2 gene is key to the process because it contributes to the synthesis of the A, B, H, Leb, and Ley antigens. Accordingly, the presence of at least one of these antigens characterizes the so-called secretor phenotype, whereas its absence, due to mutations in the enzyme’s gene coding sequence, generates the so-called nonsecretor phenotype. The secretory ABH and Lewis phenotypes of each individual depend on the combined polymorphisms at the three loci ABO, FUT2, and FUT3. Using virus-like particles (VLPs), spontaneously formed from the recombinantly expressed capsid protein of NoVs, specific recognition patterns of HBGAs have been described. This was performed by analyzing the attachment of VLPs to tissue sections of digestive tissues and to saliva mucins from HBGA phenotyped individuals, to immobilized synthetic oligosaccharides or to cells transfected in order to express the appropriate glycosyltransferases. Through these methods, it was initially noticed that A, H, and Leb antigens are ligands of the Norwalk Virus (NV), a prototype virus belonging to the GI.1 genotype (Marionneau et al., 2002). In contrast, B and Lea antigens are not recognized by this viral strain (Huang et al., 2003). Following these initial studies the glycan specificity of many other strains has been studied using VLPs belonging to all genotypes. It was observed that most strains bind to HBGAs, albeit important variations in specificities were found within each of the main human genogroups (GI and GII) (Tan and Jiang, 2011 2014). Thus, within GI two major types of binding patterns could be distinguished, that is, strains that are similar to the previously described NV GI.1 strain, recognize the A and H epitopes and strains that bind to the Lewis epitopes, targeting the α1,3/4-linked fucose residue (Fig. 3.3.1). The latter strains show variation in their ability to accommodate the α1,2-linked fucose residue and the A or B epitopes, and a dominant binding to the α1,3/4-linked fucose, allowing them to recognize nonsecretor individuals as observed by their binding to saliva from such individuals. Among GII strains three types of binding patterns have been discerned. The first one has been called ABH binder as these strains attach to saliva from all secretor individuals, regardless of the ABO and Lewis status. The second, called A/B binder, attach to saliva from secretor individuals of the A and/or B blood groups only. The third, called Lewis binder, attach to saliva of Lewis positive individuals with more or less influence of the secretor phenotype, indicating strong involvement of the α1,3/4-linked fucose (Tan and Jiang, 2011Tan and Jiang, 2014). Structural analysis of the capsid protein’s protruding region, or P-domain, cocrystallized with oligosaccharides allowed characterization of the exact binding sites for various GI and GII strains (Bu et al., 2008Cao et al., 2007Chen et al., 2011Choi et al., 2008Hansman et al., 2011). Interestingly, GI and GII binding sites appeared to be completely different, although the amino acids involved are highly conserved within each genogroup. In addition binding modes between the two genogroups are very different (Cao et al., 2007Ruvoën-Clouet et al., 2013). The GI binding sites involve contact mainly with galactose or N-acetylgalactosamine residues, while the GII sites involved essentially fucose recognition (Cao et al., 2007Koppisetty et al., 2010Nasir et al., 2012). Nevertheless, in both instances slight amino acid changes generate subtle modifications in the range of preferred oligosaccharides, allowing recognition of distinct subgroups of individuals or populations. The different ways by which GI and GII strains cope with their host’s glycan diversity are strongly suggestive of a convergent evolution and underline the important role of HBGA binding in the process of infection. These structural aspects are presented in detail in Chapter 3.1.

3. Binding of norovirus to HBGAs is involved in infection

The importance of HBGA binding in infection was first assessed through studies on volunteers. Two independent studies involving ingestion of the virus by healthy volunteers showed that nonsecretors are resistant to infection by NV (GI.1) (Hutson et al., 2005Lindesmith et al., 2003). Indeed, the challenged nonsecretor individuals presented neither clinical signs nor immune response to the infection, and the virus was absent from their feces. It was additionally observed that among individuals with the secretor phenotype, those with blood group B were not infected or remained asymptomatic, consistent with the lack of binding to the B blood group antigen by the NV strain. Thus, a combination of FUT2 and ABO alleles determines susceptibility or resistance to NV infection. More recently a new study conducted in volunteers clearly demonstrated that one GII.4 strain infected secretors almost exclusively, and regardless of their ABO phenotype (Frenck et al., 2012). Interestingly, it was recently shown in concordant studies that both secretor and Lewis status mediate susceptibility to rotavirus infection (Imbert-Marcille et al., 2014Nordgren et al., 2014Kambhampati et al., 2016).

The influence of the secretor status on susceptibility to NoV infection in authentic outbreaks was first investigated in Sweden (Thorven et al., 2005). Thirty-eight symptomatic individuals from three hospital outbreaks caused by GII.4 strains were genotyped for secretor status. In addition, the same analysis was performed on 15 symptomatic individuals from three community outbreaks caused by GI.6 and GII.6 strains. Thus, in total, 53 symptomatic and 62 asymptomatic individuals were genotyped for polymorphisms of FUT2 at nucleotides 385, 428, and 571. Strikingly, no nonsecretors were identified among the symptomatic individuals. As the secretor status was determined by genotyping, the influence of homozygosity and heterozygosity could be determined. However, no difference was identified between heterozygous and homozygous secretors. Subsequently, the resistance of nonsecretors to GII.4 strains in authentic outbreaks was confirmed by a similar study in Denmark (Kindberg et al., 2007). Similar results were also reported from a GII.4 outbreak in China (Tan et al., 2008). In that study, secretor status was determined by phenotyping of saliva samples and the weak-secretors were grouped with the secretors. In addition, two studies have investigated the influence of secretor status on susceptibility to infection with recently emerged GII.4 strains. Currier et al. (2015) investigated sporadic pediatric NoV infections and could not identify any nonsecretors among 155 patients with GII.4 infections, even though 24% of the NoV negative control individuals were nonsecretors. The GII.4 viruses identified belonged to the Den Haag 2006b (18%), New Orleans 2009 (46%), and Sydney 2012 (36%) clusters. In another pediatric material from Ecuador all 27 GII.4 single infections and all 4 coinfections that were positive for GII.4 strains were found in secretors (Lopman et al., 2015). In addition, a number of studies of NoV outbreaks caused by strains from other genogroups have reported that only secretors were identified among the symptomatically infected patients, even though the materials were not large enough to prove any statistical significance (Bucardo et al., 2009Tan et al., 2008). Furthermore, a study of Swedish blood donors showed that secretor positive individuals had significantly higher sero-prevalence and IgG antibody titers to NoV GII.4 virus than nonsecretors (Larsson et al., 2006). The remarkable match between susceptibility to infection, the presence of either A, B, or H antigens in the digestive tract and the carbohydrate specificity of the virus strongly suggested that the interaction between the virus and these glycans is an essential step of infection.

4. The complex and genotype-dependent HBGA specificities preclude a perfect match between infection and the ABO and secretor characters

However, not all strains show a clear-cut specificity for HBGA epitopes expressed by secretors as discussed earlier. Indeed, a recent study indicated that a GII.2 strain weakly binds to saliva from either A and O blood group secretors or nonsecretors Lewis positive individuals, in addition to their strong binding to saliva from B and AB secretors (Yazawa et al., 2014).

Likewise, GI.3 strains have been reported to attach to saliva from secretors as well as nonsecretors of the A, AB and O blood groups and of the Lewis positive phenotype (Shirato et al., 2008Yazawa et al., 2014). GII.7 strains bind to saliva from nearly all individuals, except Lewis negative nonsecretors (Shirato et al., 2008Yazawa et al., 2014). Besides strong binding to saliva from secretors, some binding to saliva of Lewis positive nonsecretors of a GII.6 strain was also reported (Yazawa et al., 2014). Regarding old GII.4 strains from the 1990s (US95/96), all studies indicate a strong preference for recognition of HBGAs present in secretors (Huang et al., 2005Lindesmith et al., 2008Ruvoën-Clouet et al., 2014Rydell et al., 2009b). Nonetheless, in some studies, weak binding to saliva from nonsecretor Lewis positive individuals have been observed (Ruvoën-Clouet et al., 2014). In addition, more recent strains of the GII.4 den Haag and Osaka subtypes readily attach to saliva from nonsecretors, but only if they are Lewis positive, in accordance with their ability to recognize Lewis antigens (de Rougemont et al., 2011). From these data it is to be expected that some nonsecretor individuals should be infected, albeit less frequently than secretors, and indeed this has been observed on several occasions.

Thus, a volunteers’ study performed using the GII.2 Snow Mountain Virus (SMV) strain failed to reveal an association between infection and HBGA phenotypes (Lindesmith et al., 2005), despite the fact that SMV reportedly showed strong preference for the B blood group (Harrington et al., 2002). However, that volunteers’ study was conducted on a small number of subjects, some of whom had received very high doses of virus. The volunteers’ study performed using a GII.4 strain, showed one subject of the nonsecretor phenotype that was infected, albeit presenting very mild symptoms (Frenck et al., 2012). Furthermore, a limited number of outbreaks studies also showed that nonsecretor individuals can be infected by NoV (Carlsson et al., 2009Lindesmith et al., 2005Nordgren et al., 2010Rockx et al., 2005). Both Rockx et al. (2005) and Nordgren et al. (2010) observed that GI.3 NoV can infect secretors as well as nonsecretors, in agreement with GI.3 VLP binding studies demonstrating binding of nonsecretor saliva and Lea glycoconjugates (Shirato et al., 2008Yazawa et al., 2014). Rockx et al. (2005) investigated a water-borne GI.3 NoV outbreak and found that 20/22 (91%) of the secretors and four of seven (57%) of the nonsecretors exposed to the virus became infected. Nordgren et al. (2010) investigated a food-borne GI.3 NoV outbreak, in which symptoms suggestive of NoV infection were found in seven of 15 (47%) of the nonsecretors and 26/68 (38%) of the secretors. In another study, Carlsson et al. (2009) showed that even though nonsecretor individuals had a significantly lower risk of getting infected in a GII.4 NoV outbreak in Spain, the virus also infected one individual, genotyped as a nonsecretor, suggesting that nonsecretors are not totally resistant to infections caused by the dominating NoV genotype. A similar result was obtained in a study in Burkina Faso, in which one nonsecretor and Lewis negative individual was infected with a GII.4 strain (Nordgren et al., 2013).

Although these occasional infections of nonsecretor individuals by strains such as GII.4 that show a clear preference for secretors may be explained by their ability to recognize the α1,3/4-linked fucose residue of the Lewis type present in Lewis positive (FUT3 + ) individuals even in absence of α1,2-linked fucose as in nonsecretors (FUT2−), another explanation for these cases has recently been raised. It was shown that chronic infection by virulent strains of the bacterium Helicobacter pylori aberrantly induces expression of α1,2-fucosylated motifs in the stomach mucosa of nonsecretors, which may contribute to facilitate infection of these individuals by NoV strains that otherwise show a strong preference for secretors (Ruvoën-Clouet et al., 2014).

In addition to the occasional infections of nonsecretors that seemed to contradict the early reports on the genetic susceptibility to NoV, several reports have presented apparent contradictory results concerning the association between the ABO phenotype and infection (Fretz et al., 2005Halperin et al., 2008Meyer et al., 2004Miyoshi et al., 2005). It must be noted that these studies did not take into account the strains involved and their binding specificities for HBGAs. Their interpretations are therefore dubious since, as discussed earlier, NoV strain specificities for HBGAs are variable. It is highly likely that the differences observed in NoV epidemics stem from differences in specificity for ABH antigens of the strains involved. Finally, the existence of NoVs VLPs that do not appear to attach to HBGAs has been reported (Huang et al., 2005Shirato et al., 2008Takanashi et al., 2011Yazawa et al., 2014), suggesting either the existence of human NoVs that can infect independently of HBGAs attachment or that the methods of VLPs binding characterization may not always reflect the behavior of true viruses.

5. Additional binding specificities of human norovirus

In addition to ABO(H) histo-blood group glycans, human NoV have been shown to have a number of other carbohydrate binding specificities. An early report showed that VLPs representing three different GII strains bind to a number of cell types in a heparan sulfate-dependent manner (Tamura et al., 2004). In contrast, VLPs from two GI strains only bound weakly via heparan sulfate. In addition, GII VLP have been shown to recognize sialylated glycans (Rydell et al., 2009b). In this study, VLPs from GII.4 and GII.3, but not from GI.1, were shown to recognize neoglycoproteins conjugated with SLex, SdiLex, and sialyl type-2 precursor glycans. The binding was specific as the VLPs did not bind to structural analogues including Lex and SLea. A subsequent X-ray crystal structure showed that a GII.9 NoV VLP bound SLex in the HBGA binding site (Hansman et al., 2011). In this structure, the α1,3-linked fucose was tightly bound in the fucose binding site whereas no direct contacts were identified between the protein and the sialic acid. However, for the GII.3 and GII.4 strains the sialic acid is important for the interaction as the nonfucosylated structure sialyl type-2 precursor also binds to these VLPs (de Rougemont et al., 2011Rydell et al., 2009b). Recently, binding to several additional sialylated glycans including the ganglioside GM3 was reported for GII.4 and GI.3 VLPs (Han et al., 2014). This ganglioside is widely expressed on many cell types and unlikely to confer tissue specificity. In addition, the short glycosphingolipid galactosylceramide (GalCer), organized in membrane domains and also widely expressed, has been shown to bind to human NoV (Bally et al., 2012b). Collectively, these studies indicate that charged glycans and GalCer could be involved in the NoV infection process. However, this remains to be proven, which may become possible since a culture system for human NoVs was very recently described (Jones et al., 2014) along with a novel plasmid based human NoV reverse genetic system which produces reporter virions with infectious RNA (Katayama et al., 2014).

6. Glycan-binding properties of animal noroviruses

NoV strains infecting animals also bind to glycans. Glycan binding thus appears to be a shared property within the NoV species. Thus, a recent report indicated that canine strains of the GIV and GVI genogroups recognized HBGAs expressed in the canine gut with specificities quite similar to those of the human NV strain (Caddy et al., 2014). The presence of shared ligands between humans and dogs recognized by these strains may contribute to cross-species transmission. It had previously been shown that the bovine specific GIII strains bind to the so-called alpha-Gal epitope, which is a glycan epitope synthesized by an α1,3-galactosyltransferase of the same enzyme family as the A and B blood group enzymes (Zakhour et al., 2009 2010). Humans are not able to synthesize that epitope since the corresponding GGTA glycosyltransferase gene has become a pseudogene during evolution of the apes lineage (Macher and Galili, 2008). By contrast, the alpha-Gal epitope is present in the bovine gut mucosa where it may serve as a ligand, akin to the ABH and Lewis antigens for human strains as described earlier. In this instance, the lack of expression in the human gut mucosa may confer protection from cross-species transmission.

NoV GV strains exclusively infect mice and do not appear to recognize HBGAs. Yet, they bind to glycosphingolipids of the ganglioside type. Since these strains can be cultured, it has been possible to show that the ganglioside GD1a functions as receptor for these strains (Taube et al., 2009). This is particularly interesting in view to the recent discovery that human GI and GII strains could also bind to gangliosides with affinities in the same range as those involved in HBGAs binding (Han et al., 2014).

7. Glycosphingolipids as receptors for norovirus

HBGAs are found both on glycoproteins in saliva, milk, and other body fluids, but also on membrane bound glycoproteins and glycosphingolipids (GSLs). Since glycosphingolipids, in contrast to most natural glycoproteins, only contain one glycan per molecule, they may be used to determine the precise binding specificities of glycan-binding viruses. The glycosphingolipids also, due to their amphipathic character, offer the unique possibilities to study VLP-glycan binding in dynamic membranes and not only in solution or on solid surfaces. Furthermore, such studies are motivated since glycosphingolipids have indeed been shown to function as true cellular receptors for a number of viruses (Ewers et al., 2010Neu et al., 2009Schmidt and Chiorini, 2006), including murine NoV (see earlier).

Thin-layer chromatogram binding assay (CBA) was used to show that the Norwalk virus VLP recognizes both type-1 and type-2 chain glycosphingolipids terminated with blood group A and H but not B epitopes (Fig. 3.3.2) (Nilsson et al., 2009). Subsequently, the binding of a Norwalk and Dijon (GII.4 US95/96) VLP to glycosphingolipids incorporated in fluid solid-supported lipid bilayers was studied using quartz crystal microbalance with dissipation monitoring (QCM-D) (Fig. 3.3.2) (Rydell et al., 2009a). Both VLPs recognized bilayers containing H type-1, whereas no binding was observed to bilayers containing Lea. For both VLPs, the concentration of H type-1 glycosphingolipid in the bilayer had to be above a threshold value for binding to occur, suggesting that a multivalent interaction is needed to stably attach the VLPs to the bilayer since the binding affinity of single ligands is expected to be low. Interestingly, the threshold concentration was one order of magnitude higher for the Dijon strain VLP, possibly suggesting the Norwalk strain to have a higher affinity for the H type-1 glycosphingolipid compared to the Dijon strain.


Figure 3.3.2 Detection principles of surface based glycosphingolipid binding assays with nonlabeled VLPs.
(A) Chromatogram binding assay: qualitative assay detecting VLPs bound to glycosphingolipids, separated on thin-layer chromatograms (TLC), after incubations with primary antivirus antibody and secondary enzyme-linked antibodies for visualization by substrate conversion. (B) Quartz crystal microbalance with dissipation monitoring (QCM-D). The binding kinetics of attachment to membrane associated glycosphingolipids is probed in real time based on measurements of the bound mass (∆f) and the physicochemical properties of the attached object (∆D) (Rydell et al., 2009a). (C) Total-internal-reflection fluorescence microscopy (TIRF-M). Attachment and detachment events of single vesicles, containing glycosphingolipids and a fluorescent lipid tag, interacting with VLPs bound to glycosphingolipids in a solid supported lipid bilayer. Fluorescence is registered in real time into a depth of <200 nm from the membrane due to the weak evanescent field (Bally et al., 2011). The method allows determination of the association and dissociation rates, providing a description of the complex binding kinetics (Nasir et al., 2015).

It may be calculated that the secretor α1,2 linked fucose of the H type 1 glycan cocrystallized with GII.4 VA387 (Cao et al., 2007) contributes to more than half of the glycan-protein binding energy (Koppisetty et al., 2010). Through molecular dynamics simulations, it has been suggested that human GII NoV may recognize fucosylated glycans either through secretor (α1,2 linked) or through the Lewis (α1,3/4 linked) fucose residues in the binding site (Nasir et al., 2012). The hypothesis was confirmed by crystal structures of Lewis antigens (SLex and Ley) in complex with GII NoV (Hansman et al., 2011). These so called “secretor” and “Lewis” poses (Nasir et al., 2012) could have implications in binding complex branched structures of glycoproteins or glycosphingolipids in membranes (Fig. 3.3.3).


Figure 3.3.3 Close up view of possible interactions of the NoV VA387 dimer with Lewis b glycosphingolipids.
The dimer and its ligand were obtained from the protein database id 2OBT (Cao et al., 2007) and the Lewis b glycosphingolipids were fitted, by fucose superimposition, into both the secretor and the Lewis poses at the two binding sites (Nasir et al., 2012). Although only two glycosphingolipids are expected to bind simultaneously to one dimer, the caption illustrates how different poses may affect the packing of glycosphingolipids in the membrane. In the secretor pose, with the secretor gene dependent α1,2-linked fucose (red) in the VLP binding site, the distance between the two glucose residues (purple), glycosidically linked to the ceramide moiety of glycosphingolipids is ∼ 40 Å, while in the Lewis pose, with the Lewis gene dependent α1,4-linked fucose (red) in the binding site, the distance is only ∼27 Å. A virtual membrane bilayer simulation was not available at the time of generating the illustration.

To further quantitatively characterize the virus membrane interactions and reveal the complex mathematical details of capsid protein to glycan interactions, Total Internal Reflection Fluorescent Microscopy (TIRF-M) based binding assay was recently established (Fig. 3.3.2) (Bally et al., 2011 2012a). This assay makes it possible to record the attachment and detachment events of single glycosphingolipid-containing vesicles to VLPs bound to fluid solid-supported lipid bilayers in real time (Fig. 3.3.2). Quantitative binding data describing the vesicle attachment to and detachment from Dijon (Bally et al., 2011) and Ast6139 GII.4 VLPs (Nasir et al., 2015) were obtained at both transient and steady state conditions using TIRF-M methodology.

8. Cellular uptake of norovirus

The lack of cell culture methods has made it very difficult to study NoV cell entry. However, a recent study showed replication of human NoV in cultured B lymphocytes in the presence of bacteria from the microbiota (Jones et al., 2014). The study suggested that the virus binds to HBGA expressing bacteria, which transport the virus into the cells. The significance of this mechanism for the pathogenesis of the infection remains to be determined. The low level of replication observed in cultured B cells is inconsistent with the high viral loads shed in feces of infected individuals. In addition, the mechanism does not explain the clinical resistance of nonsecretors. Thus, although they may be infected, B lymphocytes are unlikely the major cellular targets of human NoVs. In support of primary infection of epithelial cells, bovine NoV capsid protein is in the early period of the infection detected exclusively in enterocytes (Otto et al., 2011). At later time points the capsid protein is progressively detected in leucocytes of the lamina propria.

In an attempt to address the receptor function of glycosphingolipids, NoV interactions with glycosphingolipids embedded in giant unilamellar vesicles (GUVs) was studied using fluorescently labelled GII.4 VLPs and phospholipid vesicles containing glycosphingolipids and a fluorescent lipid (Rydell et al., 2013). The binding pattern to glycosphingolipids incorporated into GUVs was in full agreement with thin-layer chromatography (CBA) experiments. Upon binding to the vesicles, the VLPs induced the formation of membrane invaginations that were positive both for the fluorescent lipid and the VLP (Fig. 3.3.4). Similar invaginations have been shown to correspond to endocytosis intermediates used for cell entry for endogenous lectins, microbial toxins, viruses, and bacteria known to use glycosphingolipids for cell entry (Eierhoff et al., 2014Ewers et al., 2010Lakshminarayan et al., 2014Roemer et al., 2007). The formation of the membrane invaginations does not require clathrin or any other cytosolic coat proteins. Instead, the multivalent binding of receptor glycosphingolipids induces the formation of lipid-nanodomains that spontaneously invaginate to form tubular structures. The mechanisms by which the membrane is bent is still not clear, but likely involves line tension, asymmetric compressive stress, a specific protein–lipid geometry, and the organization of lipids in specific orientation fields in the membrane (Aigal et al., 2015Arnaud et al., 2014Johannes et al., 2014). The observation that NoV VLP has an intrinsic property to induce membrane invagination on model membranes suggests that glycosphingolipids may be functional receptors also for human NoV. Some strains of murine NoV has indeed been shown to use gangliosides as receptors on macrophages (Taube et al., 2009). When complex glycosphingolipids were depleted from the cells the infectivity of the virus was markedly decreased. Furthermore, the infectivity could be rescued by addition of the ganglioside GD1a to the cells. In addition, the entry of murine NoV has been described to be independent of caveolin and clathrin, but dependent on cholesterol (Gerondopoulos et al., 2010Perry and Wobus, 2010). These properties are characteristic also for the bacterial toxins and viruses that use the glycosphingolipid-dependent membrane invagination pathway to enter cells (Ewers et al., 2010Roemer et al., 2007).


Figure 3.3.4 Human Norovirus induces membrane invaginations upon binding to HBGA active glycosphingolipids.
Confocal image of a GII.4 VLP binding to ALeb glycosphingolipids incorporated into a giant unilamellar vesicle (Rydell et al., 2013). Scale bar, 10 μm

The apical surface of epithelial cells presents a large glycocalyx, up to 500 nm long in the duodenum that is decorated with HBGAs. It is tempting to speculate that human NoVs first attach to these motifs of the glycocalyx in order to reach their glycolipid targets that protrude a couple of nanometers only above the lipid bilayer (Fig. 3.3.2C). The HBGA-mediated glycocalyx binding would be a primary attachment and tethering step, whilst the glycosphingolipid binding would directly contribute to the entry process, as observed with the GUV experiments.

In conclusion, detailed understanding of the genetic coevolution and the cell-surface molecular interactions between enteric viruses and host receptors will hopefully contribute to the development of novel antiviral therapies and prevention of gastrointestinal infections.


Ahmed SM, Hall AJ, Robinson AE, et al. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect Dis. 2014;14:725730.

Aigal S, Claudinon J, Roemer W. Plasma membrane reorganization: A glycolipid gateway for microbes. Biochim. Biophys. Acta. 2015;1853:858871.

Arnaud J, Tröndle K, Claudinon J, et al. Membrane deformation by neolectins with engineered glycolipid binding sites. Angew. Chem. Int. Ed. 2014;53:92679270.

Bally M, Dimitrievski K, Larson G, et al. Interaction of virions with membrane glycolipids. Phys. Biol. 2012;9:026011.

Bally M, Gunnarsson A, Svensson L, et al. Interaction of single viruslike particles with vesicles containing glycosphingolipids. Phys. Rev. Lett. 2011;107:188103.

Bally M, Rydell GE, Zahn R, et al. Norovirus GII.4 virus-like particles recognize galactosylceramides in domains of planar supported lipid bilayers. Angew. Chem. Int. Ed. Engl. 2012;51:1202012024.

Bok K, Green KY. Norovirus gastroenteritis in immunocompromised patients. N. Engl. J. Med. 2012;367:21262132.

Bu W, Mamedova A, Tan M, et al. Structural basis for the receptor binding specificity of Norwalk virus. J. Virol. 2008;82:53405347.

Bucardo F, Kindberg E, Paniagua M, et al. Genetic susceptibility to symptomatic norovirus infection in Nicaragua. J. Med. Virol. 2009;81:728735.

Bucardo F, et al. Predominance of norovirus and sapovirus in Nicaragua after implementation of universal rotavirus vaccination. PLoS One. 2014;9(5):e98201.

Caddy S, Breiman A, le Pendu J, et al. Genogroup IV and VI canine noroviruses interact with histo-blood group antigens. J. Virol. 2014;88:1037710391.

Cao S, Lou Z, Tan M, et al. Structural basis for the recognition of blood group trisaccharides by norovirus. J. Virol. 2007;81:59495957.

Carlsson B, Kindberg E, Buesa J, et al. The G428A nonsense mutation in FUT2 provides strong but not absolute protection against symptomatic GII.4 Norovirus infection. PLoS One. 2009;4:e5593.

Chen Y, Tan M, Xia M, et al. Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the polymorphic human histo-blood group antigens. PLoS Pathog. 2011;7:e1002152.

Choi J-M, Hutson AM, Estes MK, et al. Atomic resolution structural characterisation of recognition of histo-blood group antigens by Norwalk virus. Proc. Natl. Acad. Sci. USA. 2008;105:91759180.

Currier RL, Payne DC, Staat MA, et al. Innate susceptibility to norovirus infections influenced by FUT2 genotype in a United States pediatric population. Clin. Infect. Dis. 2015;60:16311638.

de Rougemont A, Ruvoën-Clouet N, Simon B, et al. Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens. J. Virol. 2011;85:40574070.

Eierhoff T, Bastian B, Thuenauer R, et al. A lipid zipper triggers bacterial invasion. Proc. Natl. Acad. Sci. U S A. 2014;111:1289512900.

Ewers H, Roemer W, Smith AE, et al. GM1 structure determines SV40-induced membrane invagination and infection. Nat. Cell. Biol. 2010;12:1118.

Frenck R, Bernstein DI, Xia M, et al. Predicting susceptibility to norovirus GII.4 by use of a challenge model involving humans. J. Infect. Dis. 2012;206:13861393.

Fretz R, Svoboda P, Schorr D, et al. Risk factors for infections with norovirus gastrointestinal illness in Switzerland. Eur. J. Clin. Microbiol. Infect. Dis. 2005;24:256261.

Gerondopoulos A, Jackson T, Monaghan P, et al. Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway. J. Gen. Virol. 2010;91:14281438.

Glass RI, Parashar UD, Estes MK. Norovirus gastroenteritis. N. Engl. J. Med. 2009;361:17761785.

Gustavsson L, Andersson LM, Lindh M, et al. Excess mortality following community-onset norovirus enteritis in the elderly. J. Hosp. Infect. 2011;79:2731.

Hall A. Noroviruses: the perfect human pathogens? J. Infect. Dis. 2012;205:16221626.

Hall AJ, Eisenbart VG, Etingue AL, et al. Epidemiology of foodborne norovirus outbreaks, United States, 2001-2008. Emerg. Infect. Dis. 2012;18:15661573.

Hall AJ, Vinjé J, Lopman B, et al. Center for Disease Control and Prevention. Updated norovirus outbreak management and disease prevention guidelines. MMWR. 2011;60:115.

Halperin T, Vennema H, Koopmans M, et al. No association between histo-blood group antigens and susceptibility to clinical infections with genogroup II norovirus. J. Infect. Dis. 2008;197:6365.

Han L, Tan M, Xia M, et al. Gangliosides are ligands for human noroviruses. J. Am. Chem. Soc. 2014;136:1263112637.

Hansman GS, Biertümpfel C, Georgiev I, et al. 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. J. Virol. 2011;85:66876701.

Harrington PR, Lindensmith L, Yount B, et al. Binding of Norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice. J. Virol. 2002;76:1232512343.

Hemming M, et al. Major reduction of rotavirus, but not norovirus, gastroenteritis in children seen in hospital after the introduction of RotaTeq vaccine into the National Immunization Programme in Finland. Eur. J. Pediatr. 2013;172:739746.

Hu L, Crawford SE, Czako R, et al. Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen. Nature. 2012;485:256259.

Huang P, Farkas T, Zhong W, et al. 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. J. Virol. 2005;79:67146722.

Huang P, Xia M, Tan M, et al. Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner. J. Virol. 2012;86:48334843.

Huang PW, Farkas T, Marionneau S, et al. Norwalk-like viruses bind to ABO, Lewis and secretor histo-blood group antigens but different strains bind to distinct antigens. J. Infect. Dis. 2003;188:1931.

Hutson AM, Airaud F, Le Pendu J, et al. Norwalk virus infection associates with secretor status genotyped from sera. J. Med. Virol. 2005;77:116120.

Imbert-Marcille B-M, Barbé L, Dupé M, et al. A FUT2 gene common polymorphism determines resistance to rotavirus A of the P [8] genotype. J. Infect. Dis. 2014;209:12271230.

Johannes L, Wunder C, Bassereau P. Bending “on the rocks”—a cocktail of biophysical modules to build endocytic pathways. Cold Spring Harbor Perspect. Biol. 2014;6:a016741.

Jones MK, Watanabe M, Zhu S, et al. Enteric bacteria promote human and mouse norovirus infection of B cells. Science. 2014;346:755759.

Kambhampati A, Payne DC, Costantini V, Lopman B. Host genetic susceptibility to enteric viruses: a systematic review and metaanalysis. Clin. Infect. Dis. 2016;62:1118.

Katayama K, Murakami K, Sharp TM, et al. Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA. Proc. Natl. Acad. Sci. USA. 2014;111:E4043E4052.

Kindberg E, Akerlind B, Johnsen C, et al. Host genetic resistance to symptomatic norovirus (GGII.4) infections in Denmark. J. Clin. Microbiol. 2007;45:27202722.

Koo HL, et al. Noroviruses: the most common pediatric viral enteric pathogen at a large unviersity hospital after introduction of rotavirus vaccination. J. Pediatric Infect. Dis. Soc. 2013;2(1):5760.

Koppisetty CA, Nasir W, Strino F, et al. Computational studies on the interaction of ABO-active saccharides with the norovirus VA387 capsid protein can explain experimental binding data. J. Comput. Aided Mol. Des. 2010;24:423431.

Lakshminarayan R, Wunder C, Becken U, et al. Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers. Nat. Cell. Biol. 2014;16:595606.

Larsson MM, Rydell GE, Grahn A, et al. Antibody prevalence and titer to norovirus (genogroup II) correlate with secretor (FUT2) but not with ABO phenotype or Lewis (FUT3) genotype. J. Infect. Dis. 2006;194:14221427.

Lindesmith L, Moe C, Lependu J, et al. Cellular and humoral immunity following Snow Mountain virus challenge. J. Virol. 2005;79:29002909.

Lindesmith L, Moe CL, Marionneau S, et al. Human susceptibility and resistance to Norwalk virus infection. Nat. Med. 2003;9:548553.

Lindesmith LC, Donaldson EF, Lobue AD, et al. Mechanisms of GII. 4 norovirus persistence in human populations. PLoS Med. 2008;5:e31.

Lopman BA, Trivedi T, Vicuna Y, et al. Norovirus infection and disease in an Ecuadorian birth cohort: Association of certain norovirus genotypes with host FUT2 secretor status. J. Infect. Dis. 2015;211:18131821.

Lowe JB. The blood group-specific human glycosyltransferases. Baillieres Clin. Haematol. 1993;6:465492.

Macher BA, Galili U. The Gal〈1,3Gal®1,4GlcNAc-R (〈-Gal) epitope: A carbohydrate of unique evolution and clinical relevance. Biochem. Biophys. Acta. 2008;1780:7588.

Marionneau S, Cailleau-Thomas A, Rocher J, et al. ABH and Lewis histo-blood group antigens, a model for the meaning of oligosaccharide diversity in the face of a changing world. Biochimie. 2001;83:565573.

Marionneau S, Ruvoen N, Le Moullac-Vaidye B, et al. Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor individuals. Gastroenterology. 2002;122:19671977.

Martinez MA, Lopez S, Arias CF, et al. Gangliosides have a functional role during rotavirus cell entry. J. Virol. 2013;87:11151122.

Mesquita JR, Barclay L, Nascimento MS, et al. Novel norovirus in dogs with diarrhea. Emerg. Infect. Dis. 2010;16:980982.

Meyer E, Ebner W, Scholz R, et al. Nosocomial outbreak of norovirus gastroenteritis and investigation of ABO histo-blood group type in infected staff and patients. J. Hosp. Infect. 2004;56:6466.

Miyoshi M, Yoshizumi S, Sato C, et al. Relationship between ABO histo-blood group type and an outbreak of norovirus gastroenteritis among primary and junior high school students: results of questionnaire-based study. Kansenshogaku Zasshi. 2005;79:664671.

Nasir W, Bally M, Zhdanov VP, et al. Interaction of virus-like particles with vesicles containing glycolipids: Kinetics of detachment. J. Phys. Chem. B. 2015;119:1146611472.

Nasir W, Frank M, Koppisetty CA, et al. Lewis histo-blood group alpha1,3/alpha1,4 fucose residues may both mediate binding to GII.4 noroviruses. Glycobiology. 2012;22:11631172.

Neu U, Stehle T, Atwood WJ, The Polyomaviridae:. Contributions of virus structure to our understanding of virus receptors and infectious entry. Virology. 2009;384:389399.

Nilsson J, Rydell GE, Le Pendu J, et al. Norwalk virus-like particles bind specifically to A, H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids. Glycoconj. J. 2009;26(9):11711180.

Nordgren J, Kindberg E, Lindgren PE, et al. Norovirus gastroenteritis outbreak with a secretor-independent susceptibility pattern, Sweden. Emerg. Infect. Dis. 2010;16:8187.

Nordgren J, Nitiema LW, Ouermi D, et al. Host genetic factors affect susceptibility to norovirus infections in Burkina Faso. PLoS One. 2013;8:e69557.

Nordgren J, Sharma S, Bucardo F, et al. Both Lewis and secretor status mediate susceptibility to rotavirus infections in a rotavirus genotype dependent manner. Clin. Infect. Dis. 2014;59:15671573.

Otto PH, Clarke IN, Lambden PR, et al. Infection of calves with bovine norovirus GIII.1 strain Jena virus: an experimental model to study the pathogenesis of norovirus infection. J. Virol. 2011;85:1201312021.

Patel MM, Hall AJ, Vinje J, et al. Noroviruses: a comprehensive review. J. Clin. Virol. 2009;44:18.

Payne DC, et al. Norovirus and medically attended gastroenteritis in U.S. children. N Engl. J. Med. 2013;368(12):11211130.

Perry JW, Wobus CE. Endocytosis of murine norovirus 1 into murine macrophages is dependent on dynamin II and cholesterol. J. Virol. 2010;84:61636176.

Rockx BH, Vennema H, Hoebe CJ, et al. Association of histo-blood group antigens and susceptibility to norovirus infections. J. Infect. Dis. 2005;191:749754.

Roemer W, Berland L, Chambon V, et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature. 2007;450:670675.

Ruvoën-Clouet N, Belliot G, Le Pendu J. Noroviruses and histo-blood groups: the impact of common host genetic polymorphisms on virus transmission and evolution. Rev. Med. Virol. 2013;23:355366.

Ruvoën-Clouet N, Ganière JP, André-Fontaine G, et al. Binding of Rabbit Hemorrhagic Disease Virus to antigens of the ABH histo-blood group family. J. Virol. 2000;74:1195011954.

Ruvoën-Clouet N, Magalhaes A, Marcos-Silva L, et al. Increase in genogroup II.4 norovirus host spectrum by CagA-positive Helicobacter pylori infection. J. Infect. Dis. 2014;210:183191.

Rydell GE, Dahlin AB, Hook F, et al. QCM-D studies of human norovirus VLPs binding to glycosphingolipids in supported lipid bilayers reveal strain-specific characteristics. Glycobiology. 2009;19:11761184.

Rydell GE, Nilsson J, Rodriguez-Diaz J, et al. Human noroviruses recognize sialyl Lewis x neoglycoprotein. Glycobiology. 2009;19:309320.

Rydell GE, Svensson L, Larson G, et al. Human GII.4 norovirus VLP induces membrane invaginations on giant unilamellar vesicles containing secretor gene dependent alpha1,2-fucosylated glycosphingolipids. Biochim. Biophys. Acta. 2013;1828:18401845.

Schmidt M, Chiorini JA. Gangliosides are essential for bovine adeno-associated virus entry. J. Virol. 2006;80:55165522.

Shirato H, Ogawa S, Ito H, et al. Noroviruses distinguish between type 1 and type 2 histo-blood group antigens for binding. J. Virol. 2008;82:1075610767.

Storry JR, Olsson ML. Genetic basis of blood group diversity. Br. J. Haematol. 2004;126:759771.

Takanashi S, Wang Q, Chen N, et al. Characterization of emerging GII.g/GII.12 noroviruses from a gastroenteritis outbreak in the United States in 2010. J. Clin. Microbiol. 2011;49:32343244.

Tamura M, Natori K, Kobayashi M, et al. Genogroup II noroviruses efficiently bind to heparan sulfate proteoglycan associated with the cellular membrane. J. Virol. 2004;78:38173826.

Tan M, Jiang X. Norovirus-host interaction: Multi-selections by human histo-blood group antigens. Trends Microbiol. 2011;19:382388.

Tan M, Jiang X. Histo-blood group antigens: a common niche for norovirus and rotavirus. Expert Rev. Mol. Med. 2014;16:e5.

Tan M, Jin M, Xie H, et al. Outbreak studies of a GII-3 and a GII-4 norovirus revealed an association between HBGA phenotypes and viral infection. J. Med. Virol. 2008;80:12961301.

Taube S, Perry JW, Yetming K, et al. Ganglioside-linked terminal sialic acid moieties on murine macrophages function as attachment receptors for murine noroviruses. J. Virol. 2009;83:40924101.

Thorven M, Grahn A, Hedlund KO, et al. A homozygous nonsense mutation (428G– > A) in the human secretor (FUT2) gene provides resistance to symptomatic norovirus (GGII) infections. J. Virol. 2005;79:1535115355.

Verdaguer N, Ferrero D, Murthy MR. Viruses and viral proteins. IUCrJ. 2014;1:492504.

Vinjé J. Advances in laboratory methods for detection and typing of norovirus. J. Clin. Microbiol. 2015;53:373381.

Yazawa S, Yokobori T, Ueta G, et al. Blood group substances as potential therapeutic agents for the prevention and treatment of infection with noroviruses proving novel binding patterns in human tissues. PLoS One. 2014;9:e89071.

Zakhour M, Maalouf H, Di Bartolo I, et al. Bovine norovirus: carbohydrate ligand, environmental contamination, and potential cross-species transmission via oysters. Appl. Environ. Microbiol. 2010;76:64046411.

Zakhour M, Ruvoën-Clouet N, Charpilienne A, et al. The alphaGal epitope of the histo-blood group antigen family is a ligand for bovine norovirus Newbury2 expected to prevent cross-species transmission. PLoS Pathog. 2009;5:e1000504.

Zheng DP, Ando T, Fankhauser RL, et al. Norovirus classification and proposed strain nomenclature. Virology. 2006;346:312323.

Only gold members can continue reading. Log In or Register to continue

Stay updated, free articles. Join our Telegram channel

Apr 25, 2018 | Posted by in MICROBIOLOGY | Comments Off on Human Norovirus Receptors

Full access? Get Clinical Tree

Get Clinical Tree app for offline access