Intracellular trafficking and plasma membrane microdomain distribution of the NSP4 enterotoxin during rotavirus infection in epithelial cells
Storey, Stephen Michael
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Rotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional glycoprotein that induces secretory diarrhea in mouse pups in the absence of other viral proteins. The intracellular transport route(s) and functional mechanism(s) of NSP4 are poorly understood; however, the recent association of the enterotoxin with cellular caveolin-1 may provide a link between NSP4 transport and function. To determine if NSP4 traffics to a specific subset of lipid rafts at the plasma membrane (PM), we isolated caveolae from a PM-enriched fraction with a new method that yielded endoplasmic reticulum (ER)-free caveolae membranes with a unique membrane structure and composition. Comparison of these caveolae with other detergent- and non-detergent-extracted membranes revealed that each caveolae/raft fraction contained caveolae markers; however, only our PM caveolae fraction mimicked the membrane structure and sterol exchange dynamics of intact PM without ER or non-raft PM contaminants. When these PM caveolae were isolated from RV-infected cells, full-length, high-mannose glycosylated NSP4 was present. Confocal imaging showed association of NSP4 with caveolin-1 moving from perinuclear and cytoplasmic sites toward the PM as the infection progressed. Fluorescent imaging also indicated exposure of the NSP4 Cterminus at the exofacial PM surface without transport of the enterotoxin through the Golgi apparatus. Surface-specific biotinylation was used to confirm NSP4 exposure at the surface of infected MDCK cells and to determine that the exposed protein was fulllength and high-mannose glycosylated. This study presents an ER contaminant-free PM caveolae isolation methodology, identifies the presence of full-length, high-mannose glycosylated NSP4 in both PM caveolae and exposed at the cell surface, and confirms the Golgi-bypassing nature of NSP4 ER to PM transport in RV-infected MDCK cells.