Critical role of cellular cholesterol in bovine rotavirus infection

Abstract

Background: Bovine rotavirus (BRV) is a non-enveloped dsRNA virus that cause neonatal calf diarrhea. Lipid rafts are cholesterol-enrich membrane mircodomains that play a vital role in many cellular processes. In this study, the effect of cellular cholesterol depletion on infection of MA-104 cells with bovine rotavirus was investigated.

Results: We demonstrated that cholesterol depletion of the plasma membrane by MβCD had no effect on BRV binding to cells but significantly impaired BRV entry in a dose-dependent manner and the effect was partially reversed by addition of exogenous cholesterol, suggesting the reduction of BRV infection by MβCD was specifically due to cholesterol depletion. Cholesterol depletion after virus entry did not reduce BRV replication, whereas affected virus assembly.

Conclusions: Taken together, our results demonstrate that cell membrane cholesterol is essential to BRV infectivity.

Figure 1

Cellular cholesterol is not required for BRV attachment. (A) Cell viability after treatment with various concentrations of MβCD for 30 min was determined with CCK-8 Kit. (B) The cellular cholesterol levels were measured by Amplex Red cholesterol assay kit. (C) Cholesterol depletion had no effect on BRV attachment. Cells were mock treated or treated with indicated concentrations of MβCD for 30 min, then infected with BRV at 4°C. At 1 h later, cell lysates were prepared by freeze-thaw and virus titers were measured.

Figure 2

Cholesterol depletion reduced BRV entry. MA-104 cells were pretreated with different concentrations of MβCD followed by BRV infection for 12 h. (A) The virus titers were determined by TCID₅₀ assay. (B) Viral mRNA levels were measured by Realtime PCR, results are expressed as the fold increase relative to mock-treated control (set at 1.0). All experiments were performed three times, and the error bars indicated the standard deviations of three independent experiments. (C) After virus attachment, the unbound BRV was removed, cells were treated with MβCD at indicated time at 37°C. At 12 hpi, the levels of VP4 in the BRV-infected cells were detected by Western blotting.

Figure 3

The infection ability of BRV was partially recovered with the replenishment of exogenous cholesterol. MA-104 cells were pretreated with 10 mM MβCD for 30 min, and then supplied with 1.5 mg/ml exogenous cholesterol for 1 h followed BRV infection. (A) Content of cellular cholesterol was determined with Amplex Red cholesterol assay kit. (B) 12 h post infection, virus titers were determined. Each experiment was performed in triplicate. “***” denotes extremely significant difference in statistics (P < 0.001).

Figure 4

Cholesterol depletion after virus entry did not affect BRV replication but virus assembly. MA-104 cells were incubated with BRV for 1 h, then treated with various concentrations of MβCD, (A) viral mRNA levels (B) and virus titers were determined at 12 hpi. The results were representative of three independent experiments.