Lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle

Abstract

Lipid rafts are involved in the life cycle of many viruses. In this study, we showed that lipid rafts also play an important role in the life cycle of severe acute respiratory syndrome (SARS)-coronavirus (CoV). Cholesterol depletion by pretreatment of Vero E6 cells with methyl-beta-cyclodextrin (MbetaCD) inhibited the production of SARS-CoV particles released from the infected cells. This inhibition was prevented by addition of cholesterol to the culture medium, indicating that the reduction of virus particle release was caused by the loss of cholesterol in the cell membrane. In contrast, cholesterol depletion at the post-entry stage (3h post-infection) caused only a limited effect on virus particle release. Northern blot analysis revealed that the levels of viral mRNAs were significantly affected by pretreatment with MbetaCD, but not by treatment at 3h post-infection. Interestingly, no apparent evidence for colocalization of angiotensin converting enzyme 2 with lipid rafts in the membrane of Vero E6 cells was obtained. These results suggest that lipid rafts could contribute to SARS-CoV infection in the early replication process in Vero E6 cells.

Fig. 1

Reduction of cholesterol levels in Vero E6 cells by MβCD treatment and its effect on cell proliferation. Semi-confluent monolayers of Vero E6 cells in 6-well microplates were left untreated or were treated with 10 mM MβCD. After washing three times with PBS, the cells were collected at the indicated time points and subjected to the Amplex Red Cholesterol Assay as well as the cell proliferation assay to show the effect of MβCD on cholesterol levels (A) and cell proliferation (B), respectively. Experiments were repeated three times and the arrow bars indicate the standard deviations of three independent experiments.

Fig. 2

Reduction of virus production by MβCD treatment. Vero E6 cells were left untreated (Treat:N) or were pretreated with 5 and 10 mM of MβCD for 30 min at 37 °C (Treat:-0.5), and then infected with SARS-CoV at an MOI of 10, 0.1, or 0.001. Alternatively, cells were first infected with SARS-CoV, as described above, and then treated with MβCD for 30 min at 37 °C at 3 hpi (Treat:+3). For cholesterol replenishment, the cells were first treated with MβCD for 30 min at 37 °C, followed by replenishment of cholesterol, and then cells were infected with SARS-CoV (Treat:−0.5 + CHO). After culturing for 18 h, the virus infectivity in the culture fluids was titrated in Vero E6 cells. Experiments were repeated three times and the arrow bars indicate the standard deviations of three independent experiments.

Fig. 3

Reduction of viral mRNA levels by MβCD treatment. Vero E6 cells were left untreated (Treat:N) or were pretreated with 10 mM MβCD for 30 min at 37 °C (Treat:−0.5), and then infected with SARS-CoV at an MOI of 10. Alternatively, cells were first infected with SARS-CoV, as described above, and then treated with MβCD for 30 min at 37 °C at 3 hpi (Treat:+3). After culturing for 3 and 6 h, respectively, total RNAs were extracted and subjected to Northern blot analysis with a DNA probe for the SARS-CoV N gene. As a control for RNA input, the same amounts of the RNA samples were subjected to Northern blot analysis for GAPDH gene expression.

Fig. 4

No direct colocalization of ACE2 with lipid rafts. (A) Vero E6 cells were left untreated or were pretreated with 2.5, 5, and 10 mM MβCD for 30 min at 37 °C, and then the cells were subjected to flow cytometry analysis with antibody against ACE2. (B) Vero E6 cells before (upper four panels) or after adsorption with SARS-CoV at an MOI of 10 for 1 h at 37 °C (lower two panels) were subjected to the membrane flotation assay, as described in Section 2. After the ultracentrifugation, the samples in the tube were separated into 12 fractions. The fractions were Western-blotted and probed with antibodies against ACE2, TfR, and Cav-1. For detection of GM-1, the same fractions were dot-blotted and reacted with cholera toxin-peroxidase. Finally, these blots were visualized using the ECL Protein Detection System.