Cells and viruses.
Vero cells and B95a cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS) and were passaged twice a week. Vero cells constitutively expressing canine SLAM (Vero-SLAM cells) (49
), were cultured like Vero cells with an additional 0.5 mg/ml Zeocin. MDCK II cells were cultured in Eagle's minimal essential medium containing 5% FCS and were passaged twice a week. Two recombinant strains of CDV containing insertions of enhanced green fluorescent protein (eGPF) were used. The insertion site for eGFP in strain Onderstepoort is in front of the nucleocapsid (N) gene, while it is in front of the polymerase (L) gene in the virulent strain 5804P (41
). VSV strain Indiana, with eGFP inserted downstream of the G gene, was obtained from Gert Zimmer. The Beaudette strain of infectious bronchitis virus (IBV) was provided by Dave Cavanagh, Compton, United Kingdom. The rabbit antiserum against IBV was a kind gift from Christine Winter (51
). All viruses were propagated and plaque-titrated with 0.8% (wt/vol) methylcellulose (Sigma) in DMEM on Vero cells.
For immunofluorescence analysis, cells grown on coverslips were fixed with 3% paraformaldehyde (Merck) in phosphate-buffered saline (PBS) for 20 min at room temperature. Cells infected with viruses expressing eGFP were mounted in Mowiol (Calbiochem). Cells infected with IBV were visualized by immunostaining. For this purpose, cells were permeabilized by treatment with 0.2% Triton X-100 (Serva) in PBS for 5 min at room temperature and washed three times with 1 ml PBS. Cells were incubated with 200 μl rabbit anti-IBV (α-IBV) serum, diluted 1:200 in PBS, for 1 h at room temperature and were subsequently washed three times with 1 ml PBS. Thereafter, cells were incubated with 200 μl of fluorescein isothiocyanate-conjugated goat α-rabbit immunoglobulin G (IgG) (Sigma), diluted 1:500 in PBS, for 1 h at room temperature, washed as before, and mounted in Mowiol (Calbiochem).
Flow cytometric analysis.
For flow cytometry, 2 × 105 cells were used. Adherent cells were washed three times with 1 ml PBS, and the supernatants were collected in a centrifuge tube. To detach the cells from the bottom of the well, 200 μl trypsin was added to each well and incubated at 37°C for 10 min. Thereafter, the cells were resuspended in 1 ml PBS, added to the supernatants of the preceding washing steps, and spun down at 400 × g for 7 min at 4°C. The pellet was resuspended in 1 ml PBS, transferred to a 1.5-ml reaction tube, and centrifuged at 420 × g for 7 min at 4°C. Cells infected with a virus expressing eGFP were resuspended in 400 μl 1% paraformaldehyde (Merck) in PBS, fixed for 2 h at 4°C, and analyzed by flow cytometry. IBV-infected cells were immunostained prior to flow cytometry. For this purpose, the cell pellets were incubated for 1 h at room temperature in 200 μl rabbit α-IBV serum, diluted 1:200 in PBS. Thereafter, cells were washed three times with 1 ml PBS and centrifuged at 420 × g for 7 min at 4°C. Cells were then incubated for 1 h at room temperature in 200 μl of fluorescein isothiocyanate-conjugated α-rabbit IgG as a secondary antibody (Sigma), diluted 1:500 in PBS. After three further washing steps, the cell pellets were resuspended in 400 μl PBS each and immediately analyzed by flow cytometry. As a negative control, mock-infected cells were immunostained.
Cholesterol depletion and virus entry assays. (i) Extraction of cellular cholesterol with MβCD.
In a total volume of 1 ml DMEM containing 5% FCS, 2 × 105 Vero cells were seeded per well of a 24-well plate and incubated in a CO2 incubator overnight. Cells were washed twice with 1 ml PBS, and concentrations of methyl-β-cyclodextrin (MβCD) (stock, 250 mM in double-distilled H2O; Sigma) ranging from 0 to 15 mM, diluted in DMEM, were added to each well in a total volume of 250 μl. After incubation for 30 min at 37°C, MβCD was removed by washing the cells three times with 1 ml PBS.
(ii) Determination of cell viability after cholesterol extraction.
Cell viability was determined by propidium iodide (PI) (Sigma) staining (28
). Cells were detached from the 24-well plate by incubation with 200 μl trypsin for 10 min at 37°C. After resuspension in 1 ml PBS, they were transferred to a 1.5-ml reaction tube and pelleted at 420 × g
for 7 min at 4°C. For staining, cells were resuspended in 500 μl PBS, and 2 μg/ml PI was added. After incubation for 5 min at room temperature, the percentage of stained cells was determined by flow cytometry. Cells that were not stained with PI were used as a negative control to determine autofluorescence.
(iii) Measurement of cellular cholesterol.
To measure the effect of MβCD treatment, the cellular cholesterol content was determined using the “Cholesterin Farb Test” (Roche). A total of 2 × 107 Vero cells were seeded in a 162-cm2 cell culture flask in a total volume of 30 ml DMEM containing 5% FCS and incubated in a CO2 incubator overnight. The cell layer was washed twice with 20 ml PBS. Cells were treated either with 7.5 ml DMEM or with the same volume of DMEM containing 7.5 mM MβCD and were incubated on a rocking platform for 30 min at 37°C. Thereafter, the cells were washed three times and detached by treatment with 2 ml trypsin for 10 min at 37°C. The number of cells, resuspended in 10 ml PBS, was determined using a Thoma cell counting chamber. Equal cell numbers of both samples were spun down at 400 × g for 7 min at 4°C. The pellet was resuspended in 1 ml PBS and pelleted at 18,000 × g for 1 min at 4°C. Subsequently, the pellets were resuspended in 400 μl lysis buffer (20 mM Tris, 137 mM NaCl, 10% [vol/vol] glycerol, 1% [vol/vol] Triton X-100, 2 mM EDTA, 50 mM β-glycerolphosphate, 20 mM sodium pyrophosphate). Cell lysis was accomplished during a 20-min incubation at room temperature with vortexing of the cells every 5 min. The cholesterol content was determined, based on a colorimetric reaction, according to the manufacturer's instructions.
(iv) Infection efficiency after cholesterol depletion of cellular membranes.
Cells were either medium treated or cholesterol depleted as described above and subsequently either mock infected or infected with 250-μl virus dilutions in DMEM at multiplicities of infection (MOIs) of 0.001, 0.005, 0.01, 0.05, or 0.1, respectively, and incubated at 37°C for 1 h. After the adsorption period, cells were supplied with 1 ml methylcellulose (0.8% [wt/vol] in DMEM). Cells infected with VSV, IBV, and CDV-5804P were further processed 1 day postinfection, and cells infected with CDV-Onderstepoort were further processed 2 days postinfection, either for immunofluorescence analysis or for flow cytometry.
(v) Infection efficiency after cholesterol extraction from viral membranes.
A total of 2 × 105 Vero cells in a volume of 1 ml DMEM containing 5% FCS were seeded per well of a 24-well plate and incubated at 37°C overnight. The next day, the cell layer was washed three times with 1 ml PBS. Virus suspensions with a titer of 8 × 104 PFU/ml were treated for 30 min at 37°C with MβCD (Sigma) at concentrations ranging from 0 to 20 mM. Thereafter, 250 μl of the virus or an equal amount of medium was applied to the cell layer and incubated for 1 h at 37°C. Subsequently, cells were washed three times, overlaid with 1 ml methylcellulose (Sigma) (0.8% [wt/vol] in DMEM), and incubated as described above. Cells were processed 1 day postinfection either for immunofluorescence or for flow cytometry analysis as described before to determine the infection efficiency.
(vi) Replenishment of cholesterol.
After extraction of viral cholesterol as described above, virus was treated either with medium alone or with medium containing exogenous cholesterol (stock, 50 mM in ethanol; Sigma) at final concentrations ranging from 50 to 500 μM for 30 min at 37°C. Thereafter, cells were infected with these virus samples and prepared for immunofluorescence or flow cytometry analysis as described above.
(vii) Syncytium formation.
To study the development of CDV-induced syncytium formation in the absence of cholesterol, 2 × 105
Vero cells in 1 ml DMEM containing 5% FCS were seeded per well of a 24-well plate and incubated at 37°C overnight. The next day, the cells were washed once with 1 ml PBS, and thereafter the cells were treated with 250 μl medium or infected with 250-μl virus dilutions at an MOI of 0.1 for 1 h at 37°C. Alternatively, cells were transfected with the pCG eukaryotic expression vectors carrying the CDV-Onderstepoort H and F genes (50
) by using Lipofectamine (Invitrogen) according to the manufacturer's instructions. DNA uptake was allowed for 2 h. Subsequently, the virus inoculum or Lipofectamine/DNA, respectively, was removed by three washing steps as described before, and cellular cholesterol was extracted by treatment with 7.5 mM MβCD (Sigma) in a volume of 250 μl DMEM for 30 min at 37°C, while control cells were treated with an equal volume of medium. MβCD was removed by three washing steps as described before. To prevent replenishment of cellular cholesterol either by cholesterol uptake from the medium or by endogenous synthesis, cholesterol-depleted cells were provided with methylcellulose (Sigma) (0.8% [wt/vol] in DMEM) containing 5% delipidized FCS (PanBiotech), 10 μM mevastatin (Sigma), and 25 μM mevalonolactone (Sigma) (14
). Control cells were overlaid with methylcellulose containing 5% FCS. After incubation at 37°C, cells were fixed 1 or 2 days postinfection with 3% paraformaldehyde (Merck) in PBS and mounted in Mowiol (Calbiochem).
(viii) Quantification of syncytia.
Syncytia were quantified by counting nuclei involved in syncytia using a fluorescence microscope, taking advantage of eGFP expression. Syncytia were defined as cells containing more than three nuclei. The size of syncytia was defined by the number of nuclei per syncytium, and the amount of syncytia was determined by the number of syncytia per counting grid.
Isolation of DRMs.
A total of 3 × 107
Vero cells were seeded per 162-cm2
flask in 20 ml DMEM containing 5% FCS and incubated overnight. The next day, the cells were infected with CDV-Onderstepoort at an MOI of 0.1 in a volume of 10 ml DMEM for 1 h at 37°C. Thereafter, the inoculum was replaced by 20 ml DMEM containing 5% FCS and incubated for 1 day. Subsequently, the cell layer was washed once with PBS and incubated with 5 ml of 10 mM EDTA in PBS for approximately 1 h to detach the cells. The cell number was determined, and cells were washed twice with cold PBS. A total of 3 × 107
cells were resuspended in 300 μl MB++ buffer (MB with 100 μg/ml DNase I [Sigma, Steinheim, Germany] and 5% [vol/vol] Complete protease inhibitor cocktail [Roche, Mannheim, Germany]) (MB buffer consists of 20 mM morpholineethanesulfonic acid, 150 mM NaCl, 1% [wt/vol] Brij 98, 0.02% [wt/vol] NaN3
, pH 6.5) and kept on ice. After incubation for 16 h, the volume was adjusted to 500 μl with MB++ buffer and incubated for 5 min at 37°C. The sample was mixed with 500 μl 90% sucrose, overlaid with a linear (40 to 10%) sucrose gradient, and centrifuged in an SW41 rotor at 35,000 rpm for 20 h at 4°C. Fractions of 1 ml were collected from the top of the tube, and the pellet was resuspended in 1 ml MB+ buffer (MB++ without DNase I). Aliquots (10 μl) from each fraction were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The H and F proteins of CDV were detected by peptide-specific rabbit antisera (50
); human Lamp-2 and human flotillin-2 were detected by commercial primary antibodies (Becton Dickinson, Heidelberg, Germany). Anti-mouse IgG conjugated with horseradish peroxidase or anti-rabbit IgG conjugated with horseradish peroxidase (DAKO Cytomation, Glostrup, Denmark) served as secondary antibodies. Proteins were visualized using BM chemiluminescence blotting substrate (POD; Roche) and a ChemiDoc imager (Bio-Rad).