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 further illuminate at which step(s) cholesterol depletion reduces PRRSV replication, we first investigated the effect of cholesterol depletion on virus entry. Mock-treated, MβCD-treated and MβCD-cholesterol-treated MARC-145 cells in 24-well plates were infected with PRRSV at 10 4.5 TCID 50 per well. After incubation for 1 h, cells were harvested and intracellular PRRSV RNA levels were measured by real-time RT-PCR. As shown in Figure 3A , compared with the number of viral genomes in mock-treated cells, a significant decrease (80%) in viral genomes in MβCDtreated cells was detected. Furthermore, cholesterol replenishment partially restored intracellular viral genomes and there was only a 17% reduction after adding exogenous cholesterol. These results suggest that entry of PRRSV is dependent on the cellular membrane cholesterol. The entry phases of PRRSV infection can be divided into two stages: the first stage requires PRRSV attachment to cells, and the second stage occurs when PRRSV penetrates the cellular membrane. To determine whether MβCD reduces PRRSV attachment to MARC-145 cells, mocktreated, MβCD-treated or MβCD-cholesterol-treated MARC-145 cells were infected with PRRSV at 4°C for 1 h. All cells were collected and virus genomes were detected by quantitative real-time PCR assay. As shown in Figure 3B , virus genomes presented in MβCD-treated cells were only 23.8% of that presented in mock-treated cells. Furthermore, cholesterol replenishment partially restored virus attachment to approximately 75% of which presented in mocktreated cells, suggesting that the effect was specifically due to the cholesterol depletion ( Figure 3B ).