Unraveling the role of the secretor antigen in human rotavirus attachment to histo-blood group antigens

Abstract

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-β1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus.

Fig 1. Structure and schematic representation of the biosynthetic pathways of human type 1 and 2 histo-blood group antigens (HBGAs).

The structures the H1 and H2 antigens as well as their precursors LNB and LacNAc are shown.

Fig 2. Binding characterization of the H1 antigen and its precursor to VP8* from the P[8] genotype.

Panel a shows the affinity fit and sensorgrams of the interaction between P[8]_c and the H1 antigen obtained by SPR. Panel b shows the affinity fit and sensorgrams of the interaction between P[8]_c and LNB obtained by SPR. Panels c and d show the affinity fit and sensorgrams of the interactions between P[8]_Wa and the H1 antigen and P[8]_Wa and LNB obtained by SPR, respectively. The bars indicate the standard deviation.

Fig 3. Binding of rotavirus particles to the H1 antigen and to lacto-N-biose (LNB).

Rotavirus triple layered particles (TLPs) and double layered particles (DLPs) were used in binding assays against the H1 antigen (panel a) and against its precursor (LNB) (panel b).

Fig 4. Binding and infection blocking experiments.

Soluble lacto-N-biose (LNB) and galacto-N-biose (GNB) as well as the monosaccharides L-fucose (FUC), D-galactose, N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) were used at 20 mM to block the binding of VP8* from the P[8]_c genotype to the H1 antigen (panel a) and to LNB (panel b). The same sugars were utilized at 5 mg/mL to block the infection of Wa rotavirus in MA104 cells. Statistical differences are indicated with asterisks. * p < 0.05; **p < 0.01; ***p = 0.001 (panel c).

Fig 5. Structure of P[8]_c VP8*.

a Cartoon representation of P[8]_c VP8* Apo1 with the secondary structural element are colored in blue for β-sheets, orange for α-helices and white for loops. Different tones of blue are used to differentiate among β-strands. Secondary structural element are numbered and labeled in order from the N to C terminus. The sequence of P[8]_c VP8* is shown in panel b, highlighting with red lettering the residues that interact with H1 sugar and with blue lettering the β9-β10 hairpin. Magenta triangles indicate residues mutated in this study. Structural elements are shown above the sequence colored as in the cartoon representation.

Fig 6. Structures of P[8]_c VP8* in complex with H1 antigen and LNB.

a Cartoon representation of P[8]_c VP8* H1 structure with β-strands in different pink tones to differentiate among β-sheets, α-helices in cyan and loops in white. The H1 antigen bound between a β-sheet and the α-helix is shown in stick with carbon atoms in yellow, green and slate blue for the GlcNAc, Gal and Fuc motives. b Cartoon representation of P[8]_c VP8* LNB structure in identical orientation with β-sheets in different orange tones and α-helices in light-blue. The bound LNB is represented as in (a). c and d Close view of the P[8]_c VP8* H1 (c) and P[8]_c VP8* LNB (d) active centers. The bound sugars are represented in sticks with atoms in yellow (GlcNAc), green (Gal) and slate blue (Fuc). The residues interacting with the sugars are shown in stick representation, with carbon atoms colored according to the structural element to which they correspond (semi-transparent) and are labeled. Nitrogen and oxygen are colored in dark blue and red, respectively. Waters interacting with the sugars are represented as orange (c) and cyan (d) spheres. The mutated I173 in the back side of the sugar binding pocket is shown as stick in both panels.

Fig 7. Comparison of H1 and LNB sugar binding by P[8]c VP8*.

a Close view of the glycan binding site of superimposed P[8]c VP8* H1 (light green) and P[8]c VP8* LNB (light blue) structures (in semitransparent cartoon) showed that sugar interacting residues (in sticks and colored according to the corresponding structure) are disposed with minimal differences in comparison with the corresponding bound sugars (in sticks and colored green and stale blue for H1 and LNB, respectively). b Superimposition of the GlcNAc moieties of the H1 (in stick colored with carbons in green) and LNB (in stick colored with carbons in stale blue) from the structures of its complexes with P[8]c VP8* shows that the galactose moiety in the LNB structure occupies a more shallow position. c and d The high quality electron density maps (2Fo—Fc contoured in blue at 1σ) obtained by X-ray diffraction at 1.3 and 1.85 Å resolution for P[8]c VP8* in complex with LNB (c) and H1 (d), respectively, provides a high level of details as shown for the modelled ligands represented in sticks (carbons, oxygen and nitrogen colored in green, red and blue, respectively) and the coordinated molecules of solvent represented as red spheres inside the maps. P[8]_c VP8* proteins are shown as grey cartoons.