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

To biotinylate PRRSV, the virus was first purified following the protocols described by Nauwynck et al. [11] . The titer of the purified virus was approximately 5×10 6 TCID 50 mL 1 (50% tissue culture infectious doses mL 1 ), as determined by a cytopathic effect (CPE) assay in MARC-145 cells. The protein concentration of the virus preparation was 2 mg mL 1 , as determined by the Bradford assay with bovine serum albumin (BSA) used as a protein quantitation standard. Biotinylation was performed with a protein biotinylation kit (GE Healthcare, Uppsala, Sweden), according to manufacturer's instructions. The biotinylated viruses were collected after purification on a Sephadex G-25 column and diluted in phosphate-buffered saline (PBS; pH 7.4) at a concentration of 0.2 mg mL 1 and stored at 80°C. Virus infectivity and the ability to enter cells were not significantly decreased after being biotinylated.