Date Published: June 21, 2019
Publisher: Public Library of Science
Author(s): Roberto Gozalbo-Rovira, J. Rafael Ciges-Tomas, Susana Vila-Vicent, Javier Buesa, Cristina Santiso-Bellón, Vicente Monedero, María J. Yebra, Alberto Marina, Jesús Rodríguez-Díaz, Félix A. Rey.
Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P 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 VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P, P and P). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P 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 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 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 rotavirus.
Rotaviruses are the leading etiologic agent of viral gastroenteritis in infants and young children worldwide and are responsible for an estimated 140,000 deaths each year in developing countries . The typical classification of rotaviruses was derived from their genome composition and the immunological reactivity of three of their structural proteins: VP6, VP7 and VP4. Rotaviruses are classified into at least 7 groups (A to G) according to the immunological reactivity of the VP6 middle layer protein, with group A rotavirus being the most commonly associated with infections in human. The two outer capsid proteins VP7 and VP4, elicit neutralizing antibodies that can induce viral protection. Using these two proteins, a traditional dual classification system of group A rotaviruses into G (depending on the VP7 glycoprotein) and P (depending on the protease-sensitive VP4) types was established . At least 36 different G-serotypes and 51 P-types have been identified among human and animal rotaviruses . Viruses carrying G1[P8], G2[P4], G3[P8] and G4[P8] represent over 90% of human rotaviruses strains co-circulating in most countries , with the P genotype, which comprises four different genetic lineages (I to IV ), being particularly relevant .
Virus-HBGAs interaction has emerged as an important factor in viral infectivity. Contrarily to other enteric viruses (i.e.: norovirus), the relevance of HBGA interaction in rotaviruses was first neglected, and virus-host cell attachment studies were mainly focused on binding to sialic acid, until interactions with HBGA were suggested by VP8* structural analyses  and experimentally determined in sialidase-insensitive strains . In norovirus many studies point to the human FUT2 polymorphism as a key feature affecting viral infectivity [17, 18]. Individuals carrying two null FUT2 alleles lack fucosyl transferase-2 activity, do not express H antigen structures at the intestinal mucosa and in secretions (non-secretors) and are less susceptible to norovirus. While previous studies showed no correlation between the secretor status and rotavirus infection , the most recent studies show that antibody titers to rotavirus , rotavirus gastroenteritis incidence  and vaccine take  correlate with the FUT2 phenotype. However, the molecular mechanisms of these correlations were unknown until now. The previously reported interaction of H1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc) with the most common human rotavirus P genotype P that has been further characterized here at the structural level, highlights the importance of the secretor phenotype on the incidence of rotavirus diarrhea. We have determined the characteristics of this interaction, acknowledging a new binding site for H1 in VP8* common for all the members of P[II] genogroup. Our results show that physical interaction between the H1 antigen and P rotavirus occurs through the precursor side of the molecule (LNB), reinforcing the idea that the main carbohydrate-protein contacts are made via the N-acetyl-glucosamine moiety . NMR studies on A-antigen binding of P and P VP8*, demonstrated that the L-fucose moiety does not make contacts with VP8* and rather it remains exposed to the solvent with a high degree of flexibility . However, in the same study VP8* from genotypes P and P, that did not recognize A-antigen in our assays, were shown to bind this antigen and L-fucose-protein contacts were evidenced . Structural data from P and P VP8* in complex with LNFPI also showed a limited but direct interaction of the α1,2-linked L-fucose with the protein, namely via the R209 residue, which is conserved in all proteins from genogroup P[II] . Due to the minimal interaction of the secretory L-fucose to VP8*, the authors of this study hypothesize that this glycan moiety has a low contribution to binding affinity and that a strong interaction would be expected for the unfucosylated H1 precursor, explaining the epidemiological studies that do not correlate the FUT2 status to infection by P and P genotypes . Contrarily to this, we show that although the L-fucose moiety of H1 makes indirect contacts with P VP8*, it stabilizes the competent conformation of the LNB moiety to interact with the sugar binding residues, resulting in two-fold lower Kda for H1 compared to LNB. This small but significative difference may be of relevance in the viral susceptibility context between secretors and non-secretor (FUT2-/-) individuals. Furthermore, a weaker interaction to LNB may also explain why infection of P rotaviruses can occur, at a lower level, in non-secretor individuals  and it also accounts for the inhibitory effect of LNB in in vitro rotavirus infection reported here.