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.     

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.     

The carbohydrate moiety and high molecular weight carrier of histo-blood group antigens are both required for norovirus-receptor recognition

Histo-blood group antigens (HBGAs) on human intestinal epithelium serve as receptors for noroviruses (NVs). These antigens also are expressed in milk and may act as decoy receptors to protect breast-fed infants and others against NV disease. In this study we demonstrated that human milk is highly variable in synthesis of HBGAs, which differs from that of saliva; a large quantity of small, soluble HBGAs are found in milk, but much less in saliva and are recognized by MAbs, but not by NVs. There is another group of HBGAs, of high MW, found in both milk and saliva, and recognized by both NVs and MAbs. These results suggest that the specificity of NVs and MAbs to HBGAs are different and the backbones in addition to the carbohydrate moiety are required for NV recognition. Further studies to define the structure and genetics of the high MW milk glycans are necessary.     

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.     

Norovirus-host interaction: multi-selections by human histo-blood group antigens

The discovery of human histo-blood group antigens (HBGAs) as receptors or ligands of noroviruses (NoVs) raises a question about the potential role of host factors in the evolution and diversity of NoVs. Recent structural analysis of selected strains in the two major genogroups of human NoVs (GI and GII) demonstrated highly conserved HBGA binding interfaces within the two groups but not between them, indicating convergent evolution of GI and GII NoVs. GI and GII NoVs are probably introduced to humans from different non-human hosts with the HBGAs as a common niche. Each genogroup has further diverged into multiple sub-lineages (genotypes) through selections by the polymorphic HBGAs of the hosts. An elucidation of such pathogen-host interaction, including determination of the phenotypes of NoV-HBGAs interaction for each genotype, is important in understanding the epidemiology, classification and disease control and prevention of NoVs. A model of this multi-selection of NoVs by HBGAs is proposed.     

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.     

Crystal structures of GI.8 Boxer virus P dimers in complex with HBGAs,a novel evolutionary path selected by the Lewis epitope

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le^b) and Le^y antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the β-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.     

Antigenic Relatedness of Norovirus GII.4 Variants Determined by Human Challenge Sera

The GII.4 noroviruses (NoVs) are a single genotype that is responsible for over 50% of NoV gastroenteritis epidemics worldwide. However, GII.4 NoVs have been found to undergo antigenic drifts, likely selected by host herd immunity, which raises an issue for vaccine strategies against NoVs. We previously characterized GII.4 NoV antigenic variations and found significant levels of antigenic relatedness among different GII.4 variants. Further characterization of the genetic and antigenic relatedness of recent GII.4 variants (2008b and 2010 cluster) was performed in this study. The amino acid sequences of the receptor binding interfaces were highly conserved among all GII.4 variants from the past two decades. Using serum samples from patients enrolled in a GII.4 virus challenge study, significant cross-reactivity between major GII.4 variants from 1998 to 2012 was observed using enzyme-linked immunosorbent assays and HBGA receptor blocking assays. The overall abilities of GII.4 NoVs to bind to the A/B/H HBGAs were maintained while their binding affinities to individual ABH antigens varied. These results highlight the importance of human HBGAs in NoV evolution and how conserved antigenic types impact vaccine development against GII.4 variants.     

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.     

Histo-blood group antigens act as attachment factors of rabbit hemorrhagic disease virus infection in a virus strain-dependent manner

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50-90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level.     

Structural basis for the recognition of blood group trisaccharides by norovirus

Noroviruses are one of the major causes of nonbacterial gastroenteritis epidemics in humans. Recent studies on norovirus receptors show that different noroviruses recognize different human histo-blood group antigens (HBGAs), and eight receptor binding patterns of noroviruses have been identified. The P domain of the norovirus capsids is directly involved in this recognition. To determine the precise locations and receptor binding modes of HBGA carbohydrates on the viral capsids, a recombinant P protein of a GII-4 strain norovirus, VA387, was cocrystallized with synthetic type A or B trisaccharides. Based on complex crystal structures observed at a 2.0-A resolution, we demonstrated that the receptor binding site lies at the outermost end of the P domain and forms an extensive hydrogen-bonding network with the saccharide ligand. The A and B trisaccharides display similar binding modes, and the common fucose ring plays a key role in this interaction. The extensive interface between the two protomers in a P dimer also plays a crucial role in the formation of the receptor binding interface.     

Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.     

A Unique Human Norovirus Lineage with a Distinct HBGA Binding Interface

Norovirus (NoV) causes epidemic acute gastroenteritis in humans, whereby histo-blood group antigens (HBGAs) play an important role in host susceptibility. Each of the two major genogroups (GI and GII) of human NoVs recognizes a unique set of HBGAs through a distinct binding interface that is conserved within a genogroup, indicating a distinct evolutionary path for each genogroup. Here, we characterize a Lewis a (Le^a) antigen binding strain (OIF virus) in the GII.21 genotype that does not share the conserved GII binding interface, revealing a new evolution lineage with a distinct HBGA binding interface. Sequence alignment showed that the major residues contributing to the new HBGA binding interface are conserved among most members of the GII.21, as well as a closely related GII.13 genotype. In addition, we found that glycerol inhibits OIF binding to HBGAs, potentially allowing production of cheap antivirals against human NoVs. Taken together, our results reveal a new evolutionary lineage of NoVs selected by HBGAs, a finding that is important for understanding the diversity and widespread nature of NoVs.     

Binding Patterns of Rotavirus Genotypes P[4], P[6], and P[8] in China with Histo-Blood Group Antigens

Rotaviruses (RVs) are an important cause of severe gastroenteritis in children. It has been found that RV may recognize the histo-blood group antigens (HBGAs) as ligands or receptors and bind HBGAs in a type-dependent manner. In this study, we investigated the binding specificity of VP8* proteins from human rotaviruses (RV) that are prevalent in China including genotypes P[4], P[6], and P[8]. Through the saliva- and oligosaccharide-based binding assays, we found that the VP8* proteins of P[4] and P[8] RV showed similar reactivity with the Le^b and H type 1 antigens, while P[6] RV weakly bound the Le^b antigen. These findings may facilitate further research into RV host specificity and vaccine development.     

组织血型抗原:轮状病毒可能的潜在受体

轮状病毒(RVs)是引起婴幼儿及动物非细菌性胃肠炎的重要病原体.研究发现,人类组织血型抗原(HBGAs)可能是RVs的结合受体.HBGAs具有丰富的多态性,包含ABO、分泌型及Lewis抗原.研究表明,不同P型的RVs与HBGAs的结合具有型特异性,而且不同人群对各型RVs易感性存在差异.因此,研究RVs与HBGAs的相互作用对于阐明RV感染的致病机理及RV疫苗的设计具有重要意义.     

G Ⅱ.23基因型诺如病毒P蛋白的寡糖结合特征

诺如病毒(Noroviruses,NoVs)是导致人急性胃肠炎的最重要病原体之一,也是引起食源性疾病暴发的首要病原体。组织血型抗原(Histo-blood groups antigens,HBGAs)是NoVs的受体或宿主易感因子。已有研究表明HBGAs与NoVs的感染和流行高度相关。GⅡ.23是最近报道的NoVs新基因型。为了研究GⅡ.23与HBGAs的结合特征,表达纯化GⅡ.23基因型的P蛋白之后,通过唾液和寡糖结合实验研究其与HBGAs的结合特性,并通过同源结构模拟探索GⅡ.23 P蛋白与糖抗原潜在的对接分子机制,与已经解析的GⅡ.10的P蛋白与岩藻糖的复合物结构进行重叠。结果发现,GⅡ.23 P蛋白可以与B型唾液结合,但不结合A、O^+和O^-非分泌型唾液;P蛋白与H双糖抗原发生结合;分子模拟显示GⅡ.23 P蛋白具有与岩藻糖环结合的类似特征。本研究首次揭示了GⅡ.23 P蛋白与HBGAs受体的结合特征,为深入探索GⅡ.23基因型NoVs的进化、感染以及流行的具体机制提供了基础资料。     

Interaction between noroviruses and human histo-blood group antigens

Norovirus (NOV), a member of the family Caliciviridae, is a major cause of water and food-borne acute nonbacterial gastroenteritis, and forms many morphologically similar but antigenically diverse groups of viruses. The virus-like particles (VLPs) derived from the prototype strain of NoV, Norwalk virus (NV/68), bind to histo-blood group antigens (HBGAs). HBGAs are carbohydrates that contain structurally related saccharide moieties, and are found in saliva and mucosal secretions from intestinal epithelial cells of secretor individuals who have FUT2 gene encoding a fucosyltransferase. From volunteer challenge studies, there is strong evidence that the carbohydrate-binding is essential for the NV/68 infection. Non-secretors, who do not express FUT2 fucosyltransferase and consequently do not express H type 1 or Leb in the gut, were not infected after the challenge with NV/68. However, other NoV VLPs display different ABH and Lewis carbohydrate-binding profiles, and indeed epidemiological studies showed that some NoV strains could infect individuals with another ABH phenotypes. GII/4 is known to be global epidemic strain and bound more HBGAs when compared with other strains. The strength of the transmission of GII/4 strains may be linked with their wide recognition of HBGAs. It is obvious that HBGAs are important factors to determine the host specificity, although it is still unclear whether the HBGAs act as the primary receptor or enhance NoV infectivity. Further investigation is needed.     

Evolution of the interactions between GII.4 noroviruses and histo-blood group antigens: Insights from experimental and computational studies

Norovirus (NoV) is the major pathogen causing the outbreaks of the viral gastroenteritis across the world. Among the various genotypes of NoV, GII.4 is the most predominant over the past decades. GII.4 NoVs interact with the histo-blood group antigens (HBGAs) to invade the host cell, and it is believed that the receptor HBGAs may play important roles in selecting the predominate variants by the nature during the evolution of GII.4 NoVs. However, the evolution-induced changes in the HBGA-binding affinity for the GII.4 NoV variants and the mechanism behind the evolution of the NoV-HBGA interactions remain elusive. In the present work, the virus-like particles (VLPs) of the representative GII.4 NoV stains epidemic in the past decades were expressed by using the Hansenula polymorpha yeast expression platform constructed by our laboratory, and then the enzyme linked immunosorbent assay (ELISA)-based HBGA-binding assays as well as the molecular dynamics (MD) simulations combined with the molecular mechanics/generalized born surface area (MMGBSA) calculations were performed to investigate the interactions between various GII.4 strains and different types of HBGAs. The HBGA-binding assays show that for all the studied types of HBGAs, the evolution of GII.4 NoVs results in the increased NoV-HBGA binding affinities, where the early epidemic strains have the lower binding activity and the newly epidemic strains exhibit relative stronger binding intensity. Based on the MD simulation and MMGBSA calculation results, a physical mechanism that accounts for the increased HBGA-binding affinity was proposed. The evolution-involved residue mutations cause the conformational rearrangements of loop-2 (residues 390–396), which result in the narrowing of the receptor-binding pocket and thus tighten the binding of the receptor HBGAs. Our experimental and computational studies are helpful for better understanding the mechanism behind the evolution-induced increasing of HBGA-binding affinity, which may provide useful information for the drug and vaccine designs against GII.4 NoVs.     

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.