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. 2011 May;85(9):4057-70.
doi: 10.1128/JVI.02077-10. Epub 2011 Feb 23.

Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens

A de Rougemont  1 , N Ruvoen-Clouet, B Simon, M Estienney, C Elie-Caille, S Aho, P Pothier, J Le Pendu, W Boireau, G Belliot
Affiliations

Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens

A de Rougemont et al. J Virol. 2011 May.

Abstract

Noroviruses (NoVs) are one of the leading causes of gastroenteritis in children and adults. For the last 2 decades, genogroup II genotype 4 (GII.4) NoVs have been circulating worldwide. GII.4 NoVs can be divided into variants, and since 2002 they have circulated in the population before being replaced every 2 or 3 years, which raises questions about the role of their histo-blood group antigen (HBGA) ligands in their evolution. To shed light on these questions, we performed an analysis of the interaction between representative GII.4 variants and HBGAs, and we determined the role of selected amino acids in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the amino acids, directly implicated in interactions with HBGAs. However, the ablation of the threonine residue at position 395 (ΔT395), an epidemiological feature of the post-2002 variants, was not deleterious to the binding of the virus-like particle (VLP) to the H antigen, while binding to A and B antigens was severely hampered. Nevertheless, the ΔT395 VLPs gained the capacity to bind to the Lewis x and sialyl-Lewis x antigens in comparison with the wild-type VLP, demonstrating that amino acid residues outside the HBGA binding site can modify the binding properties of NoVs. We also analyzed the attachment of baculovirus-expressed VLPs from six variants (Bristol, US95/96, Hunter, Yerseke, Den Haag, and Osaka) that were isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs. We showed that the six variants could all attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, Den Haag and Osaka variants additionally bound to carbohydrates present in the saliva of Lewis-positive nonsecretors. The carbohydrate binding profile and the genetic and mutagenesis analysis suggested that GII.4 binding to Lewis x and sialyl-Lewis x antigens might be a by-product of the genetic variation of the amino acids located in the vicinity of the binding site. Analysis of the binding properties for the six variants by surface plasmon resonance showed that only post-2002 variants (i.e., Hunter, Yerseke, Den Haag, and Osaka) presented strong binding to A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post-2002 variants. The combination of increased affinity for ABH antigens and of a newly acquired ability to recognize glycans from Lewis-positive nonsecretors could have contributed to the epidemiological importance of strains such as the Den Haag GII.4 subtype.

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Figures

Fig. 1.
Fig. 1.
Characterization of the purified VLPs of the GII.4 variants. (A) Drawing to scale of the amino acid residues (S343, T344, R345, D373, S442, G443, and Y444) from the α-fucose binding site (white arrowhead) (8, 49) and the inserted threonine residue, T395 (black arrowhead). The NH2 terminus of the P1 domain is truncated. (B) SDS-PAGE analysis of the VLPs from the GII.4 variants (lanes 2 to 7) and the mutagenized Hunter VLPs (lanes 9 through 21). The name of the NoV strain is indicated in parentheses for each variant. The mutagenized VLPs originated from the Hunter variant (strain E1057), and the location of the amino acid residues is based upon the ORF2 amino acid sequence of the E1057 strain (GenBank accession number EU876890). Two micrograms of bovine serum albumin (Pierce) was added as a control for the protein estimation (lane 8). Lane 1 contains the protein molecular size markers that are indicated on the right side of the gel. (C) Atomic force microscopy imaging of the Hunter purified VLPs. The aggregates are shown in white. One of the VLPs is indicated by a bracket. Scale bar, 250 nm. (D) Size distribution of the VLPs as determined from atomic force microscopy imaging. The variant VLPs are color coded according to the legend on the right side of the graph. (E) Electron micrograph of the CsCl-purified Hunter VLPs after negative staining. Complete and subunit VLPs are indicated by arrows with and without a tail, respectively.
Fig. 2.
Fig. 2.
Comparison of the binding of the mutagenized VLPs Y444F and ΔT395 with the wt Hunter VLPs. (A) The VLPs were tested in duplicate on a panel of HSA glycoconjugates, and the mean values are plotted on the graph. Ordinate and abscissa indicate the optical density at 450 nm (OD450) and the nature of the glycoconjugate, respectively. The legend is at the right of the graph. (B) Relative binding of the Y444F, ΔT395, and wt Hunter VLPs for the LNFP-I (H type 1 pentasaccharide) and A and B BSA conjugates. LNFP-I and A and B trisaccharide conjugates were diluted 2-fold in carbonate/bicarbonate buffer, pH 9.6, from 103 to 0.15 μg per well. The amount of coated neoglycoconjugate is indicated in nanograms per well (abscissa). For each variant and neoglycoconjugate, the mean results of triplicate binding experiments and standard deviations are shown (vertical bars). Binding values are given by the absorbance at 450 nm (ordinate). The glycoconjugate used for each assay is indicated on the graph (abscissa). A-tri, A trisaccharide; B-tri, B trisaccharide; Le, Lewis; Sec, secretor; Non-sec, nonsecretor.
Fig. 3.
Fig. 3.
Saliva binding assays of the GII.4 variants. The binding experiments were performed in duplicate for each sample, and the mean values are given on the graph (OD450; ordinate). The Lewis status is indicated on the abscissa by minus (absence of Lewis antigen) and plus (presence of Lewis antigen) signs. The nonsecretor (non sec) and HBGAs are also indicated on the abscissa and are separated by dashed lines. The type of VLP is indicated on the right side of the graph.
Fig. 4.
Fig. 4.
Binding of the GII.4 variant VLPs to a panel of neoglycoconjugates. The binding experiments were performed in duplicate for each HSA glycoconjugate, and the mean values are plotted on the graph. Ordinate and abscissa indicate the OD at 450 nm and the nature and the origin of the oligosaccharide moiety, respectively. Details about the neoglycoconjugates are shown in Table 3. Variants are identified by the legend at right, with the name of the isolate in parentheses.
Fig. 5.
Fig. 5.
Relative binding affinity of the GII.4 NoV variants for the LNFP-I (H type 1 pentasaccharide) and A and B BSA conjugates by ELISA (A, C, and E) and SPR (B, D, and F). The H antigen (A and B), A antigen (C and D), and B antigen (E and F) BSA conjugates used for the ELISA and SPR analysis originated from the same stock. The neoglycoconjugates are indicated above each graph. The ELISA binding assay was similar to that described for the Hunter mutated VLPs (Fig. 2B). For each variant and neoglycoconjugate, mean results of triplicate binding experiments and standard deviations are shown (vertical bars). Binding values are given by the absorbance at 450 nm (ordinate). For the SPR binding assay, the same VLP and neoglycoconjugate preparations were used as for the ELISA. The response (ordinate) is given in resonance units (RU). The sensorgrams are color coded as indicated on the graphs.
Fig. 6.
Fig. 6.
MST of the GII.4 variants. The number of sequences for each group of variants is indicated in parentheses. Identical sequences are represented by gray-shaded circles, which are scaled according to member count. The white and light gray circles represent one and two sequences, respectively. For dark gray and black circles, the number of identical sequences is indicated inside each circle. The six GII.4 isolates, which were analyzed during the study, are indicated by red circles. Each group of variants is color coded according to the date of circulation, as described previously (2). For the six variants that were analyzed, the binding profile to secretor and nonsecretor HBGAs is summarized in Table 4. For the A, B, and H antigens, the size of the letter is scaled according to the numbers of RU that were determined during the SPR analysis in the experiment shown in Fig. 5.
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