Canine distemper virus infection requires cholesterol in the viral envelope

Abstract

Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-beta-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the viral envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the viral envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested.

FIG. 1.

Effect of cholesterol depletion of Vero cells on CDV-Onderstepoort infection. (A and B) Effect of cholesterol depletion of Vero cells by increasing MβCD concentrations on CDV infection efficiency. Vero cells were depleted of cholesterol by increasing MβCD concentrations for 30 min at 37°C and were subsequently infected with CDV-Onderstepoort (squares), VSV (circles), or IBV (triangles). VSV and IBV samples were prepared for quantification by flow cytometry (A) or fluorescence microscopy (B) 1 day postinfection, and CDV-Onderstepoort samples were prepared 2 days postinfection. Infection rates without MβCD obtained by flow cytometry were set to 100%. Results are means from three independent experiments with standard deviations. Asterisks indicate significance (P < 0.05) by the t test. (C) Effect of cholesterol depletion of Vero cells by 7.5 mM MβCD on CDV-Onderstepoort, IBV, or VSV infection at different MOIs. Vero cells were either treated with medium or cholesterol depleted by treatment with 7.5 mM MβCD and were subsequently infected with increasing MOIs of CDV-Onderstepoort, IBV, or VSV. Quantification was done by flow cytometry. Results are means from three independent experiments with standard deviations. Asterisks indicate significance (P < 0.05) by the t test. (D) Vero cells were treated with medium or 7.5 mM MβCD prior to infection with CDV-Onderstepoort, IBV, or VSV at an MOI of 0.001. Plaque numbers were counted and set to 100% for untreated samples. Results are means from three independent experiments with standard deviations. Asterisks indicate significant values (P < 0.05) by the t test.

FIG. 2.

Effect of cholesterol depletion of Vero-SLAM cells on infection with CDV-5804P. (A) CDV-5804P infection of Vero cells and Vero-SLAM cells. Vero cells and Vero-SLAM cells were infected at an MOI of 0.1 and fixed 1 day postinfection. Infection was monitored by fluorescence microscopy. Results are representative of three independent experiments. (B) Infection efficiency of CDV-5804P for cholesterol-depleted Vero-SLAM cells. Vero-SLAM cells were treated with medium or 7.5 mM MβCD prior to infection with CDV-5804P at an MOI of 0.1, 0.01, or 0.001. Infection efficiencies were monitored by fluorescence microscopy. Results are representative of three independent experiments.

FIG. 3.

Role of CDV-Onderstepoort envelope cholesterol in infection. (A) Infection efficiency of cholesterol-depleted CDV on Vero cells. Envelope cholesterol of CDV or VSV was depleted by treatment with increasing MβCD concentrations, and these viruses were subsequently used to infect Vero cells. The number of infected cells was measured by flow cytometry 1 day postinfection. Infection rates without MβCD were set to 100%. Results are means from three independent experiments with standard deviations. Significant values (P < 0.05) by the t test are indicated by asterisks for CDV and by number signs (#) for VSV. (B) Infection efficiency of cholesterol-depleted CDV after replenishment with exogenous cholesterol. CDV was cholesterol depleted by treatment with 5 mM MβCD. Prior to infection of Vero cells, cholesterol was replenished by increasing concentrations of exogenous cholesterol. Infection efficiencies were determined by flow cytometry 1 day postinfection. Infection rates for control virions, treated neither with MβCD nor with cholesterol, were set to 100%. Results are means from three independent experiments with standard deviations. Asterisks indicate significant values (P < 0.05) by the t test.

FIG. 4.

Effect of cholesterol depletion of cells during the course of CDV infection on syncytium formation. (A) Syncytium formation of CDV grown on Vero cells under cholesterol starvation conditions. Vero cells either were infected with CDV or VSV or were mock infected as a control. One hour postinfection, cells were cholesterol depleted by incubation with MβCD following treatment with mevastatin and mevalonolactone and addition of delipidized FCS to prevent replenishment of cholesterol. Alternatively, cells were medium treated and subsequently provided with a medium containing normal FCS. VSV-infected cells were fixed and prepared for immunofluorescence 1 day postinfection; CDV-infected cells were prepared 2 days postinfection. Images are representative of three independent experiments. (B) Quantification of syncytium formation following CDV-Onderstepoort infection. The size and number of syncytia were determined following CDV-Onderstepoort infection of Vero cells grown normally or under cholesterol starvation conditions during the course of infection. Syncytia were defined as cells with more than three nuclei. The size of syncytia was defined as the number of nuclei per cell and the amount as the number of syncytia per counting square. Results are means from five counting grids from three independent experiments, with standard deviations. Asterisks indicate significant (P < 0.05) values by the t test.

FIG. 5.

Distribution of the CDV envelope proteins in DRMs. CDV-infected Vero cells were lysed in 1% Brij 98 and subjected to linear sucrose gradient centrifugation. Aliquots of the gradient and pellet (P) fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CDV proteins H and F as well as the cellular proteins Lamp-2 and flotillin-2 were detected by Western blot analysis.