Heterogeneity of Raft-type membrane microdomains associated with VP4, the rotavirus spike protein, in Caco-2 and MA 104 cells.
Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-beta-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.
Delmas
Olivier
O
INSERM UMRS 538, Faculty of Medicine Pierre et Marie Curie, site Saint Antoine, University Pierre and Marie Curie, 27 rue de Chaligny, 75012 Paris, France.
Breton
Michelyne
M
Sapin
Catherine
C
Le Bivic
André
A
Colard
Odile
O
Trugnan
Germain
G
D000818:Animals; D018938:Caco-2 Cells / Q000378:metabolism / Q000821:virology; D036022:Capsid Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006028:Glycosphingolipids / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D010743:Phospholipids / Q000032:analysis / Q000302:isolation & purification; D011485:Protein Binding / Q000187:drug effects; D012401:Rotavirus / Q000502:physiology*; D013045:Species Specificity; D019065:Virus Assembly; D047392:beta-Cyclodextrins / Q000494:pharmacology
D036022:Capsid Proteins; D006028:Glycosphingolipids; D010743:Phospholipids; C060339:VP4 protein, Rotavirus; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin