Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition.
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 degrees 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.
Rolsma
M D
MD
Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana 61801.
Gelberg
H B
HB
Kuhlenschmidt
M S
MS
D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005678:G(M2) Ganglioside / Q000494:pharmacology; D005679:G(M3) Ganglioside / Q000494:pharmacology; D005732:Gangliosides / Q000302:isolation & purification / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000382:microbiology; D009439:Neuraminidase / Q000494:pharmacology; D011991:Receptors, Virus / Q000302:isolation & purification / Q000378:metabolism*; D012401:Rotavirus / Q000378:metabolism*; D013552:Swine
D005679:G(M3) Ganglioside; D005732:Gangliosides; D011991:Receptors, Virus; D005678:G(M2) Ganglioside; D009439:Neuraminidase