STRUCTURE AND FUNCTION OF A GANGLIOSIDE RECEPTOR FOR PORCINE ROTAVIRUS

A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and s howed to specifically inhibit the binding of porcine rotavirus (OSU st rain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschm idt, J. Virol. 68:258-268, 1994), was further purified and found to co ntain two major monosialogangliosides. Each ganglioside was purified t o apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperomet ric detection and fast atom bombardment mass spectroscopy. Both gangli osides possessed a sialyllactose oligosaccharide moiety characteristic of GM(3) gangliosides, Compositional analyses indicated that each gan glioside was composed of sialic acid, galactose, glucose, and sphingos ine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. An N-glycol ylneuraminic acid (NeuGc) moiety was found in the more polar porcine G M(3), whereas the less polar GM, species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM(3), anti NeuAcGM(3) displayed dose-dependen t inhibition of virus binding to host cells. NeuGcGM(3), was approxima tely two to three times more effective than NeuAcGM(3), in blocking vi rus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM(3)/50 ng of virus (similar to 2 x 10(7) virions) and 2 x 1 0(6) cells/ml, Fifty percent inhibition of binding was achieved with 0 .64 and 1.5 mu M NeuGcGM(3), and NeuAcGM(3),, respectively. The free o ligosaccharides 3'- and 6'-sialyllactose inhibited binding 50% at mill imolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition, Di rect binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM(3), and NeuAcGM(3), was demonstrated by using a thin-layer chromatographic ove rlay assay, NeuGcGM(3) and NeuAcGM(3), inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9.84 mu M, respectiv ely. NeuGcGM(3), (700 nmol/g [dry weight] of intestine) was found to b e the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM(3), (200 nmol/g [dry weight] of intestine). NeuGcGM(3) and NeuAcGM(3), tog ether comprised nearly 100% of the lipid-bound sialic acid in the neon atal intestine, but their quantities rapidly diminished during the fir st 5 weeks of life, These data support the hypothesis that porcine Neu GcGM(3), and NeuAcGM(3) are physiologically relevant receptors for por cine rotavirus (OSU strain). Further support for this hypothesis was o btained from virus binding studies using mutant or neuraminidase-treat ed cell lines, Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Le c-1 cells, a mutant CHO clone characterized by a deficiency in glycosy lation of N-linked oligosaccharides, still bound rotavirus, Furthermor e, exogenous addition of NeuGcGM(3), to the Lec-2 mutant cells restore d their ability to bind rotavirus in amounts equivalent to that of the ir parent (CHO) cell line. In the virus-permissive MA-104 cell line, N euGcGM(3), was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides pla y a major role in recognition of host cells by porcine rotavirus (OSU strain).