Cholesterol of lipid rafts is a key determinant for entry and post-entry control of porcine rotavirus infection

Xiujing Dou, Yang Li, Junlan Han, Dante S. Zarlenga, Weijuan Zhu, Xiaofeng Ren, Na Dong, Xunliang Li, Guangxing Li

Lipid rafts are major structural components in plasma membranes that play critical roles in many biological processes including virus infection. However, few reports have described the relationship between lipid rafts and porcine rotavirus (PRV) infection. In this study, we investigated whether or not the locally high concentrations (3-5 fold) of cholesterol present in lipid rafts are required for PRV infection, and further examined which stages of the infection process are most affected. When cellular cholesterol was depleted by methyl-β-cyclodextrin (MβCD), PRV infectivity significantly declined in a dose-dependent manner. This inhibition was partially reversed upon reintroduction of cholesterol into the system. This was corroborated by the co-localization of PRV with a recombinant, GPI-anchored green fluorescent protein, which functioned as a marker for membranous regions high in cholesterol and indicative of lipid rafts. Changes in virus titer and western blot analyses indicated that depletion of cellular cholesterol with MβCD had no apparent effect on PRV adsorption; however, depletion of cholesterol significantly restricted entry and post-entry of PRV into the cell. Both points of inhibition were restored to near normal levels by the addition of exogenous cholesterol. We conclude from these studies that membrane-based cholesterol and in particular that localized to lipid rafts, is an indispensable biomolecule for PRV infection, and that cholesterol-based control of the infection process takes place during entry and immediately post-entry into the cell.