Non-lytic extraction and characterisation of receptors for multiple strains of rotavirus.

To characterise the cellular receptors for rotavirus, we used the detergent octyl-beta-D-glucopyranoside (octyl-glucoside/OG) to extract the receptors for bovine, simian, porcine and human rotaviruses from MA104 and HT29 cells. An octyl-glucoside concentration of 0.2% dramatically reduced the susceptibility of treated cells to infection, while leaving them metabolically active, and as a result the depleted receptors were able to regenerate. Periodate treatment of the MA104 and HT29 octyl-glucoside extracts significantly decreased the ability of these extracts to neutralise rotavirus infectivity, revealing carbohydrate as component of the extracted receptors for Wa and NCDV. Treatment of MA104 cells with the metabolic inhibitors tunicamycin, deoxymannojirimycin and BenzylGalNAc suggested N-linked carbohydrate may be more important than O-linked in infection by some strains of rotavirus. Furthermore, by including cycloheximide during the regeneration of depleted receptors we found evidence that porcine rotavirus CRW8 may use a glycolipid-based receptor, while NCDV and Wa use a glycoprotein. The regenerating properties of the rotavirus receptors allowed repeated harvesting of cell surface molecules using octyl-glucoside on consecutive days, and these extracts were used to visualise virus binding in a virus overlay protein blot assay (VOPBA). Using VOPBAs, we observed both Wa and NCDV appear to recognise proteins of approximately the same molecular weight present on MA104 and HT29 cells.

Rotavirus infection of MA104 cells is inhibited by Ricinus lectin and separately expressed single binding domains.

Various lectins were tested for blocking rotavirus infection of MA104 cells and it was observed that galactose-specific lectins were the most inhibitory. Of these Ricinus agglutinin was able to inhibit infection (by human and animal strains) at concentrations as low as 10(-9) M. In addition, in a virus overlay protein blot assay Ricinus agglutinin competed with simian rotavirus SA11 for binding to solubilized MA104 proteins. Amino acid sequence comparisons revealed similarity between the ricin toxin B subunit (which contains two separate carbohydrate-binding motifs: single binding domains (SBD) 1 and 2) and rotavirus spike protein VP4. A filamentous phage display system was used to independently express the two binding domains and while SBD1 inhibited infection of MA104 cells by CRW8, NCDV, and to a lesser extent Wa, SBD2 blocked only CRW8 and NCDV infection. Furthermore inhibition of CRW8 infection was a direct result of phage inhibiting virus attachment to cells. When amino acid 248 within SBD2 was mutated from the ricin toxin to the Ricinus agglutinin sequence this phage clone showed reduced binding to galactose and was no longer able to inhibit virus infection. Thus, rotavirus recognizes galactose as an important component of the receptor on MA104 cells.