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.
Jolly
C L
CL
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia. rota@microbiology.unimelb.edu.au
Beisner
B M
BM
Ozser
E
E
Holmes
I H
IH
D000818:Animals; D002417:Cattle; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology; D003902:Detergents; D005960:Glucosides; D019073:HT29 Cells; D006801:Humans; D008934:Mitogens / Q000494:pharmacology; D010504:Periodic Acid; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011991:Receptors, Virus / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D013045:Species Specificity; D013552:Swine; D013997:Time Factors; D014764:Viral Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism
D002457:Cell Extracts; D003902:Detergents; D005960:Glucosides; D008934:Mitogens; D011500:Protein Synthesis Inhibitors; D011991:Receptors, Virus; D014764:Viral Proteins; D010504:Periodic Acid; C018619:octyl-beta-D-glucoside; D003513:Cycloheximide; C009288:metaperiodate