Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition

Abstract

A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100°C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.

Sialic Acids in Molecular and Cellular Interactions

“…Sialidase-sensitive binding and its inhibition by glycoproteins such as glycophorin A or fetuin have also been demonstrated for a porcine group C rotavirus (Svensson, 1992). Glycolipids can serve as potential receptors for a porcine group A rotavirus (Rolsma et al, 1994) because two monosialoglycolipid fractions revealed sialidase-sensitive inhibition of virus interaction with porcine enterocytes. However, defined structures for these glycolipids have not been described.…”

“…Instead, several different glycoconjugates have been reported to mediate binding of one or the other strain or to function as inhibitors. Regarding Sias, binding to sialylated glycoconjugates has been found for simian (Bass et al, 1991;Chen et af., 1993;Willoughby, 1993) and for porcine (Rolsma et al, 1994;Svensson, 1992) strains. Simian rotavirus (group A) binds specifically to highmolecular-weight mucins prepared from suckling mice, whereas much lower binding was observed with mucins from adult mice and, using the same experimental approach, no such molecule could be detected in rats, which are not infected by this rotavirus (Bass et al, 1991).…”

Evolution of Cell Recognition by Viruses: A Source of Biological Novelty with Medical Implications

“…Gangliosides (Rolsma et al, 1994(Rolsma et al, , 1998; integrins 2 1 (VLA-2) (Coulson et al, 1997;Ciarlet et al, 2002) Negative-sense, single-stranded RNA viruses (Gavrilovskaya et al, 1998(Gavrilovskaya et al, , 1999) Filoviridae…”

Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo

“…Binding of parental and reassortant rotavirus strains to cultured cells treated with neuraminidase. Previous studies (16,21,28,35) demonstrated that the decreased infectivity produced by neuraminidase treatment was caused by decreased viral binding to cells. To ensure that the effect of neuraminidase on each of our reassortants was due to changes in viral binding, an assay to measure the amount of infectious virus bound to treated and untreated MA-104 cells was performed (1).…”