Functional And Structural Characterization Of Group A Rotavirus And Group C Rotavirus

Objective To explore the glycan-binding specificity of the VP8~* protein of porcine group-C rotavirus strain(Por2011);To speculate the receptor binding site of Por2011 and to provide a certain basis for understanding the transmission of animal RVCs;To investigate the receptor binding characteristics of human G6P[8] Rotateq VP8~* protein and to analyze the structural basis of Rotateq VP8~* protein interacting with glycans and explore the receptor binding mechanism of P[8] VP8~* proteins;To study the receptor binding characteristics of bovine G1P[5] VP8~* protein,and the crystal structure of P[5] VP8~* protein was analyzed to provide a basis for exploring the interaction mechanism between P[5] RVA and glycans.Methods(1)The VP8~* gene sequence of G6P[5] RVC Por2011 was obtained from Gen Bank database and cloned into plasmid p GEX4T-1.Por2011 GST-VP8~* protein was expressed and purified.The glycan binding characteristics were s tudied and verified by functional experiments.Sequence alignment and mutation analysis identified the potential receptor binding sites.(2)The VP8~* gene sequence of G6P[8] strain Rotateq P[8] was obtained from Gen Bank database for gene synthesis and cloned into the expression vectors p GEX4T-1 and p ET-30 a,respectively;The GST-VP8~* protein and VP8~*-His protein was expressed and purified.P[8] BEL,P[8] Lab,P[4],P[6] GST-VP8~* proteins were also obtained simultaneously;The receptor binding characteristics of Rotateq P[8] GST-VP8~* protein were studied by functional assays.The Rotateq P[8] VP8~* protein was crystallized by sitting drop vapor diffusion method,and then co-crystallized with core2 and H1.The crystal data of P[8] VP8~* protein were collected by X-ray diffraction.The glycan-binding site of P[8] VP8~* was compared with the binding sites of P[4],P[6] and P[19] VP8~* proteins.(3)The VP8~* gene sequence of G1P[5] was obtained from Gen Bank database for gene synthesis,cloned into the expression vectors p GEX4T-1 and p ET-30 a respectively.The GST-VP8~* protein and His-tagged VP8~* protein was expressed and purified;The glycan binding specificity of bovine P[5] VP8~* was characterized by enzyme immunoassay and Biolayer Interometry(BLI)analysis;Crystallization screening of the His-tagged VP8~* protein was carried out using the sitting-drop vapor diffusion method,and the crystal data of P[5] VP8~* protein was obtained by X-ray diffraction.Results(1)Por2011 GST-VP8~* protein was successfully expressed and purified.Functional assay showed that Por2011 VP8~* protein presented relatively high binding to the glycans containing P1 antigen(Gal?1-4Gal?1-4Glc Nac?).The point mutation of the amino acid 107 of por2011 VP8~* protein showed it was crucial for the interaction with P1 antigen.(2)VP8~* proteins were successfully expressed and purified.P[8] VP8~* protein bound to mucin core 2 and H1 antigens.The residues W81,L167,Y169,G170,G171,R172,W174,T185,R209 and E212 of P[8] VP8~* protein participated in the interaction with core2 to form the glycan binding site.The binding site of LNFP1 is the same as that of core2.(3)The bovine P[5] GST-VP8~* protein and VP8~*-His protein were expressed and purified;The bovine P[5] RV GST-VP8~* protein exhibited obvious binding to several glycans containing sialic acid;The bovine P[5] GST-VP8~* structure was solved,showing a typical galectin-like structure,and the structural superimposition exhibited that P[5] VP8~* was most close to the human Wa P[8] VP8~*.Conclusion(1)Some porcine RVCs may use P1 antigen as a potential receptor,and the receptor binding site is closer to that of human RVCs,which provides a basis for the study of the infection mechanism of porcine RVCs.(2)Mucin core2 and H1 antigens may serve as important cell attachment factors of P[8] RVAs.P[8] VP8~* possed the same glycan binding site as VP8~* proteins of P[4] P[6] and P[19].(3)G1P [5] RV can interact with a variety of sialic acid glycans and may infect host cells through sialic acid;The crystal structure of bovine P[5] VP8~* is closest to P[8] VP8~*,however,the amino acids of the putative sialic acid binding site were quite different to those of P[3]/P[7] VP8~* and showed various conformation.These results implied that P[5] may bind to sialic acid using different interaction mechanism or glycan binding sites.