Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells

Sun, Ying; Xiao, ShaoBo; Wang, Dang; Luo, Rui; Li, Bin; Chen, HuanChun; Fang, LiuRong

Confluent MARC-145 cells in 24-well plates were incubated with 10 μg of biotinylated PRRSV in PBS per well at 4°C for 1 h to achieve the maximal virus-cell attachment. Supernatants were removed and the cells were washed three times with cold PBS. Cells were next left untreated, or treated with MβCD, or treated with MβCD and cholesterol replenishment, as described above. During the treatment, viruses complete the penetration process. After removal of the supernatant, cells were incubated in DMEM containing 2% FBS for 1 h, and then washed three times with cold PBSA and incubated with 1:50-diluted streptavidin-fluorescein isothiocyanate (FITC) for 1 h on ice. Subsequently, cells were washed once with PBS and resuspended in cold PBS containing 4% paraformaldehyde and fixed for 10 min at room temperature. After removal of paraformaldehyde, cells were washed twice with PBS containing 2% FBS, and the biotinylated virions were visualized on a confocal laser scanning microscope (LSM 510 Meta; Carl Zeiss, Gottingen, Germany).