Molecular basis of P[II] major human rotavirus VP8* domain recognition of histo-blood group antigens.

Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Le(b)) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Le(b) (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an alpha-helix and a beta-sheet, referred as the "beta alpha binding site", the P[8] and P[4] VP8*s bind Le(b) HBGAs in a previously undescribed pocket formed by the edges of two beta-sheets, referred to as the "beta beta binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Le(b) type 1 HBGAs using the beta alpha binding site. The presence of two distinct binding sites for Le(b) and non-Le(b) HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics. Author summary Rotaviruses (RV)s are the main cause of severe diarrhea in humans and animals. Significant advances in understanding RV diversity, evolution and epidemiology have been made after discovering that RVs recognize histo-blood group antigens (HBGAs) as host cell receptors or attachment factors. While different RV strains are known to have distinct binding preferences for HBGA receptor ligands, their molecular basis in controlling strain-specific host ranges remains unclear. In this study, we used solution nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the molecular-level details for interactions of the human P[8] and P[6] RV VP8* domains with their HBGA receptors ligands. The distinct binding patterns observed between these major human RVs and their respective glycan ligands provide insight into the evolutionary relationships between different P[II] genotypes that ultimately determine host ranges, disease burden, zoonosis and epidemiology, which may impact future strategies for development of vaccines to protect against RV infections.