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-Å 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.

BackgroundHuman noroviruses are the major viral pathogens of epidemic acute gastroenteritis. These genetically diverse viruses comprise two major genogroups (GI and GII) and approximately 30 genotypes. Noroviruses recognize human histo-blood group antigens (HBGAs) in a diverse, strain-specific manner. Recently the crystal structures of the HBGA-binding interfaces of the GI Norwalk virus and the GII VA387 have been determined, which allows us to examine the genetic and structural relationships of the HBGA-binding interfaces of noroviruses with variable HBGA-binding patterns. Our hypothesis is that, if HBGAs are the viral receptors necessary for norovirus infection and spread, their binding interfaces should be under a selection pressure in the evolution of noroviruses.Methods and FindingsStructural comparison of the HBGA-binding interfaces of the two noroviruses has revealed shared features but significant differences in the location, sequence composition, and HBGA-binding modes. On the other hand, the primary sequences of the HBGA-binding interfaces are highly conserved among strains within each genogroup. The roles of critical residues within the binding sites have been verified by site-directed mutagenesis followed by functional analysis of strains with variable HBGA-binding patterns.Conclusions and SignificanceOur data indicate that the human HBGAs are an important factor in norovirus evolution. Each of the two major genogroups represents an evolutionary lineage characterized by distinct genetic traits. Functional convergence of strains with the same HBGA targets subsequently resulted in acquisition of analogous HBGA binding interfaces in the two genogroups that share an overall structural similarity, despite their distinct locations and amino acid compositions. On the other hand, divergent evolution may have contributed to the observed overall differences between and within the two lineages. Thus, both divergent and convergent evolution, as well as the polymorphic human HBGAs, likely contribute to the diversity of noroviruses. The finding of genogroup-specific conservation of HBGA binding interfaces will facilitate the development of rational strategies to control and prevent norovirus-associated gastroenteritis.

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 Ley or sialyl Lex 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.