Structural Basis of Rotavirus Strain Preference toward N-Acetyl- or N-Glycolylneuraminic Acid-Containing Receptors

The rotavirus spike protein domain VP8 star is essential for recognition of cell surface carbohydrate receptors, notably those incorporating N-acylneuraminic acids (members of the sialic acid family). N-Acetylneuraminic acids occur naturally in both animals and humans, whereas N-glycolylneuraminic acids are acquired only through dietary uptake in normal human tissues. The preference of animal rotaviruses for these natural N-acylneuraminic acids has not been comprehensively established, and detailed structural information regarding the interactions of different rotaviruses with N-glycolylneuraminic acids is lacking. In this study, distinct specificities of VP8 star for N-acetyl- and N-glycolylneuraminic acids were revealed using biophysical techniques. VP8 star protein from the porcine rotavirus CRW-8 and the bovine rotavirus Nebraska calf diarrhea virus (NCDV) showed a preference for N-glycolyl-over N-acetylneuraminic acids, in contrast to results obtained with rhesus rotavirus (RRV). Crystallographic structures of VP8 star from CRW-8 and RRV with bound methyl-N-glycolylneuraminide revealed the atomic details of their interactions. We examined the influence of amino acid type at position 157, which is proximal to the ligand's N-acetyl or N-glycolyl moiety and can mutate upon cell culture adaptation. A structure-based hypothesis derived from these results could account for rotavirus discrimination between the N-acylneuraminic acid forms. Infectivity blockade experiments demonstrated that the determined carbohydrate specificities of these VP8 star domains directly correlate with those of the corresponding infectious virus. This includes an association between CRW-8 adaption to cell culture, decreased competition by N-glycolylneuraminic acid for CRW-8 infectivity, and a Pro157-to-Ser157 mutation in VP8 star that reduces binding affinity for N-glycolylneuraminic acid.