Functional Analysis of Interactions of Rotavirus NSP4 with Caveolin-1, Cyclophilin A, Cyclophilin 40, Heat Shock Protein 56, and Cholesterol

The rotavirus enterotoxin, NSP4, is responsible for early secretory diarrhea associated with rotavirus infection and is critical for RV replication and morphogenesis. NSP4 interacts directly with caveolin-1 and cholesterol and traffics from the endoplasmic reticulum to the plasma membrane via an unconventional transport pathway for release prior to virus-induced lysis. In this study we demonstrate that NSP4 interacts with the immunophilins, cyclophilin A, cyclophilin 40, and HSP56 using co-immunoprecipitation and FRET analysis. We examined the roles of caveolin-1 and the immunophilins in NSP4 transport to the plasma membrane using silencing RNA, immunofluorescence analysis, and surface biotinylation. Cholesterol reduction was accomplished by statin inhibition of cholesterol synthesis. We found that knockdown of these cellular proteins altered the intracellular distribution of NSP4, but did not prevent NSP4 from trafficking to the PM. Cholesterol inhibition decreased the amount of NSP4 that reached the PM, indicating a role of cholesterol in NSP4 transport