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

Abstract

bThe rotavirus spike protein domain VP8* 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* for N-acetyl-and N-glycolylneuraminic acids were revealed using biophysical techniques. VP8* 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* 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* 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* that reduces binding affinity for N-glycolylneuraminic acid.

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

“…The VP8* domain of the RV outer capsid spike protein VP4 mediates this process (44), but the identity of VP8* receptors is quite controversial. It was believed that VP8* recognized either terminal sialic acid or internal sialic acid, mainly based on crystallographic and NMR studies (45)(46)(47)(48). However, recently a human strain (HAL1166) with a P [14] VP8* was found to bind to A-type histo-blood group antigen (49), a neonatal strain with a P [11] VP8* bound to type 2 precursor glycans (50), and several other P types recognized secretorrelated antigens Lewis b and H type 1 (51).…”

Crystallographic and Glycan Microarray Analysis of Human Polyomavirus 9 VP1 Identifies N -Glycolyl Neuraminic Acid as a Receptor Candidate

“…In all three cases, two hydrogen bonds are formed between protein residues and the extra hydroxyl group in Neu5Gc. Mutation of any of these residues results in a significant loss of activity of CRW-8 VP8 or SubB (35,39). In contrast to HPyV9, the binding site for Neu5Gc is more surface exposed in CRW-8 VP8 and STEC SubB, with one side of the ring facing the protein and the other side facing the solvent.…”

“…Similar to HPyV9 VP1, CRW-8 VP8 can bind both Neu5Ac and Neu5Gc, but it prefers the latter compound, which shows a higher binding affinity and results in increased cellular infectivity (39). Likewise, SubB can also bind Neu5Ac and Neu5Gc in glycan microarrays, but structural studies were successful only for the Neu5Gc complex, again indicating a preference for Neu5Gc binding (35).…”

“…Although sialic acids often display modifications, the molecular principles that govern recognition of sialic acid variants are poorly understood, as the majority of structures have been solved using Neu5Ac-based compounds as ligands. Neu5Gc, which can be synthesized by nonhuman mammals but not by humans, has attracted much interest in recent years due to its role in understanding and defining host and tissue tropism (35,38,39). In order to investigate differences in sialic acid receptor binding in two closely related viruses from different species, we analyzed the ligand specificity and determined the structure of the major capsid protein VP1 from HPyV9, which was isolated from humans, and compared its properties with those of the VP1 protein from the monkey-derived virus LPyV.…”

“…Similarly, animal rotavirus strains have variable preferences for Neu5Gc or Neu5Ac. The porcine and bovine rotaviruses prefer Neu5Gc as a component of their attachment receptor, whereas the rhesus monkey rotavirus apparently utilizes Neu5Ac-type receptors for infection (39). Finally, the subtilase cytotoxin secreted by Shiga-toxigenic Escherichia coli (STEC) specifically recognizes Neu5Gc-type glycan receptors in the human gut epithelium and kidney vasculature (35).…”