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.

Curcumin inhibits bovine herpesvirus type 1 entry into MDBK cells

“…A number of studies have identified curcumin as a membrane-modulating agent, for example, it could alter lipid bilayer properties and influence structurally and functionally unrelated proteins, including a wide range of membrane proteins (Ingolfsson et al, 2007(Ingolfsson et al, , 2014, We have previously reported that cellular membrane lipid raft is crucial in mediating BoHV-1 entry into MDBK cells (Zhu et al, 2010). Here we demonstrated that curcumin affected BoHV-1 entry into MDBK cells.…”

“…The collected viruses were aliquoted, titrated and stored at - Cytotoxicity assay. Monolayers of MDBK cells seeded in 96-well plates were incubated with curcumin at concentrations 5, 10, 20 and 40 μmol/l at 37 o C for 24 hr, and the cytotoxicity was determined by MTT reduction assay as previously described (Zhu et al, 2010). The mean optical density of the control wells treated with DMSO was assigned as a value of 100%.…”

“…After treatment with citric buffer and extensive washing with phosphate-buffered saline (PBS), DMEM medium (400 μl) with or without chemicals was added to each well. After 24 hr of incubation, the cells were subjected to two freeze-thaw cycles, and the virus yield was titrated by TCID 50 assay (Zhu et al, 2010). To evaluate whether serum affected the antiviral activity of curcumin, the experiment was performed similarly except that 2% horse serum was added to DMEM.…”

Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin–TLR5 signaling

“…We used MβCD to deplete cholesterol from cell membranes (Simons and Toomre 2000;Vercauteren et al 2010;Zhu et al 2010). Methyl-β-cyclodextrin (MβCD) cytotoxicity to Caco-2 and IPEC-J2 cells was evaluated using the Cell Proliferation Reagent WST-1 (Roche, Germany).…”

“…A monolayer of 1 × 10 5 cells/well in a 96-well tissue culture plate (Corning) was pre-incubated with 2 mM MβCD for 1 h, and where indicated, supplemented with 400 μg/ml cholesterol (Sigma, USA) for 1 h at 37 °C (Zhu et al 2010). Cells were washed gently three times with PBS, and then 1 × 10 7 CFUs of bacterial strains were added to each well.…”