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25 Páginas, 7 figuras, 2 tablas ; 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. ; This work was supported by Spanish Government (Ministerio de Economia y Competitividad) grants AGL2014-52996-C2-2-R and RYC-2013-12442 to JRD, AGL2014-52996-C2-1-R to MJY, BIO2016-78571-P to AM and by Valencian Government grant Prometeo II/2014/029 to AM. RGR is the recipient of a postdoctoral grant from the Valencian Government APOST/2017/037, JRCT is the recipient of fellowship FPU13/02880 from Ministerio de Educación, Cultura y Deporte, SVV is recipient of a predoctoral fellowship from Valencian Government ACIF/2016/437. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Peer reviewed