Critical role of cholesterol in bovine herpesvirus type 1 infection of MDBK cells

Abstract

Cholesterol is involved in the life cycle of many viruses. Here, we examined the role of cholesterol for both viral envelope and target cell membrane for bovine herpesvirus type 1 (BoHV-1) infection. Cholesterol depletion by pretreatment of Madin-Darby bovine kidney (MDBK) cells with a cholesterol-sequestering drug methyl-beta-cyclodextrin (MbetaCD), inhibited the production of BoHV-1 in a dose-dependent manner. This inhibitory effect was partially reversed by cholesterol replenishment, indicating that the reduction was caused by cholesterol depletion. Cholesterol depletion at the post-entry stage only had a mild effect on the virus production. However, cell membrane cholesterol depletion did not reduce the virus attachment. In addition, treatment of BoHV-1 particles with MbetaCD also reduced the virus infectivity significantly and the effect was partially reversed by addition of exogenous cholesterol. Taken together, these data implicated that cell membrane cholesterol mainly contributed to BoHV-1 entry into MDBK cells and the viral envelope cholesterol was also essential for the virus infectivity.

Fig. 1

Depletion of cholesterol in MDBK cells by MβCD and the effect on cell proliferation. (a) Cell viability after 30 min treatment with MβCD at various concentrations was determined using the MTT method, with the number of viable cells being expressed as a % of mock treated cells. Experiments were repeated three times, and the error bars indicate the standard deviations of three independent experiments. (b) Depletion of cholesterol from the membrane of MDBK cells was measured by detecting CTB-FITC binding to GM1 of the cells. Cells were mock treated or treated with MβCD at various concentrations. CTB binding occurred for 30 min on ice and was measured by FACS where cellular autofluorescence (without CTB-FITC) was used as the control. Experiments were repeated twice, and the error bars indicate the standard deviations of two independent experiments.

Fig. 2

Cholesterol depletion reduced BoHV-1 entry (a) and the inhibitory effect was partially reversed by exogenous cholesterol (b). (a) Productive BoHV-1 entry into MDBK cells treated with MβCD at various concentrations was measured by plaque formation assay. Mock treated cells were used as a control. (b) MβCD treated cells were treated with 400 μg/ml cholesterol for 1 h before BoHV-1 infection. After treatment with citric buffer, the productive BoHV-1 entry was measured by plaque formation assay. Mock treated cells and the cells treated with MβCD but not with cholesterol were used as controls. Experiments were repeated three times, and the error bars indicate the standard deviations of three independent experiments.

Fig. 3

Cholesterol depletion at the post-entry stage mildly affected the virus yield. MDBK cells were first infected with BoHV-1, 1 h post-inoculation followed by treatment with citric buffer they were then treated with 10 mM MβCD for 30 min (Treat: +1). In the controls cells were mock treated (Treat: mock) or pretreated with 10 mM MβCD for 30 min (Treat: −0.5), and then infected with BoHV-1, followed by treatment with citric buffer. The virus production was measured by plaque formation assay. Experiments were repeated three times, and the error bars indicate the standard deviations of three independent experiments.

Fig. 4

Cholesterol depletion did not reduce attachment of BoHV-1. (a) Binding of BoHV-1 to the target cells was determined with virus titration. MDBK cells were mock treated or were treated with 5 and 10 mM MβCD for 30 min, and then binding of BoHV-1 to the cells was processed at 4 °C for 1h. The amount of cell-associated virus was titrated in MDBK cells. (b) Binding of BoHV-1 to the target cells was determined with FACS. Cells were mock treated or treated with 10 mM MβCD and then infected or mock infected with BoHV-1 at 4 °C. At 1h later, the cells were fixed with 4% paraformaldehyde and stained with bovine anti-BoHV-1 serum followed by FITC-conjugated anti-bovine immunoglobulin. Experiments were repeated three times, and the error bars indicate the standard deviations of three independent experiments. Image is one of three independent experiments.

Fig. 5

Viral envelope cholesterol was required for the virus infectivity. (a) Infectivity of BoHV-1 which was mock treated or treated with various concentrations of MβCD, was measured by plaque formation assay. (b) Infectivity of BoHV-1 which was replenished with exogenous cholesterol was measured by plaque formation assay. Cell was first mock pretreated or pretreated with 10 mM MβCD and then mock replenished or replenished with 400 μg/ml cholesterol for 1 h before BoHV-1 infection. Experiments were repeated three times, and the error bars indicate the standard deviations of three independent experiments.