Plasmids used for this work - Hybridisation of CDV-H, -F or -M with eGFP

C.7 PCR-protocols

C.7.4 Hybridisation of CDV-H, -F or -M with eGFP

2.3 Plasmids used for this work

2.3 Plasmids

Table 2.3:Plasmids used for this work

Inserted gene Organism of origin Plasmid Source

F CDV-Onderstepoort (Small²) pCG1 Veronika von Messling, INRS, Canada (von Messling et al., 2001) F CDV-Onderstepoort (Small²) pCG1-Zeocin Veronika von Messling,

INRS, Canada

H CDV-Onderstepoort (Large³) pCG1 Veronika von Messling, INRS, Canada (von Messling et al., 2001) H CDV-Onderstepoort (Large³) pCG1-Zeocin Veronika von Messling,

INRS, Canada

M CDV-Onderstepoort pcDNA3.1 Veronika von Messling,

INRS, Canada eGFP Aequorea victoria, modulated pEGFP-N1 Clontech

- - pCG1 Veronika von Messling,

INRS, Canada

- - pCG1-Zeocin Veronika von Messling,

INRS, Canada

1MDCK = Madin Darby Canine Kidney

2Small plaque forming strain

3Large plaque forming strain

3. Methods

3.1 Cell culture

3.1.1 Cell maintenance and passage

All cell lines used showed adherence and were either cultured in 25 cm²-flasks with a total volume of 5 ml medium or in 75 cm²-flasks in a total volume of 20 ml medium. Cells were split when reaching confluency, normally every three to four days. Therefore, the medium was discarded and the cells were washed once with 5 or 10 ml PBSM, respectively. Subsequently, to detach the cells, they were incubated with 0.5 or 1 ml trypsin at 37^◦C for around 10 min, respectively. Detached cells were resuspended in 4.5 or 9 ml fresh medium, respectively, split 1:20 and provided with fresh medium including the required additives. All Vero cell lines were maintained in Edulb containing 5 % FCS, MDCK I and MDCK II cells were maintained in EMEM containing 5 % FCS, Calu-3 cells were maintained in EMEM containing 5 % FCS and 1 % sodium pyruvate and B95a cells were maintained in Edulb with 5 % FCS. Cells were cultivated in an CO2-incubator at 37^◦C in 5 % CO2 atmosphere.

3.1.2 Isolation of primary canine epithelial cells of the respiratory tract

The lung of a beagle from an unrelated experiment was dissected. For transport the lung was stored in sterile PBS. The trachea and the upper bronchi were prepared and incubated in digestion-medium for 24 h at 4^◦C. Thereafter, trachea and bronchi were opened longitudinally and the epithelial cells lining the inner trachea and bronchi were detached by scraping them off with a scalpel and subsequently they were taken up in PBS. Cells were pelleted for 10 min

52 3. Methods

at 190 g and resuspended in an appropriate volume of incubation medium. To remove larger pieces of tissue, cells were passed through a 100µm cell filter before seeding into a cell culture flask and incubated in a CO2-incubator overnight (o/n). The other day, medium was changed to remove unsettled cells. The first passage was used for experiments, after reaching confluency.

3.1.3 Freezing and thawing of cells

Cells were trypsinised from the bottom of the flask as described for cell passaging and pelleted at 400 g for 7 min at room temperature. Around 5 x 10⁶ cells were resuspended in 1 ml cell freezing medium. After resuspension cells were quickly frozen at -80^◦C.

All cells except Vero-SLAM cells were thawed at 37^◦C, quickly diluted in 30 ml cold PBS and pelleted at 400 g for 7 min at room temperature. The pellet was resuspended in 20 ml medium containing the required additives and cultured in a cell culture flask with 75 cm² growth area in an CO2-incubator. Vero-SLAM cells were thawed at 37^◦C, quickly added to medium in a cell culture flask and allowed to attach to the bottom of the flask for 3 h at 37^◦C. Thereafter, to remove residual DMSO, the medium was carefully exchanged with fresh one.

3.1.4 Determination of cell numbers

Cells were counted in a cell counting chamber, model Thoma new.

3.2 Viruses

3.2.1 Propagation

4 x 10⁶ Vero cells (Vero-SLAM cells for CDV-5804P) were seeded in a 75 cm²cell culture flask in a total volume of 15 ml Edulb containing 5 % FCS and incubated at 37^◦C. The next day, after the cell layer had reached around 90 % confluency, the cells were infected at an MOI between 0.05 and 0.1 in a total volume of 5 ml Edulb for 1 h at 37^◦C. Thereafter, the volume was adjusted to 15 ml Edulb with 5 % FCS and the cells were incubated as before.

3.2. Viruses 53

Virus was harvested when most of the cells were infected, but not yet detached from the flask.

For VSV this was usually the case after 12 - 16 hours and for CDV-Onderstepoort, CDV-5804P as well as for IBV between one and two days.

For harvesting IBV and all CDV strains, the cells were scratched into the medium with a cell scraper. Hepes buffer was added before the cell suspension was frozen and thawed two times in liquid nitrogen and at 37^◦C to release cell-associated virus into the medium. Cell debris was pelleted at 3 000 g for 10 min at 4^◦C. Aliquots of the supernatant were frozen in liquid nitrogen and stored at -80^◦C.

VSV was harvested after removal of cell debris by centrifugation at 3 000 g for 10 min at 4^◦C.

Aliquots of the supernatant were frozen in liquid nitrogen.

3.2.2 Plaque test

2 x 10⁵Vero cells (Vero-SLAM for CDV-5804P) were seeded in 1 ml Edulb containing 5 % FCS per well of a 24-well plate. The cells were incubated at 37^◦C o/n. The next day, the cells had reached 100 % confluency. A 10-fold dilution series (10⁻¹- 10⁻⁵ for IBV and all CDV strains;

10⁻¹- 10⁻⁷ for VSV) in Edulb was prepared. The cell layer was washed once with 1 ml PBS per well and 250µl virus dilution was added to each well. As a control, 250µl medium were added to the mock-infected well and cells were incubated for one hour in at 37^◦C. Thereafter 1 ml methylcellulose in Edulb containing 5 % FCS and standard antibiotics was added to each well and incubated as before.

VSV and IBV were fixed 1 dpi, CDV-5804P 2 dpi, and CDV-Onderstepoort 3 dpi with 3 % PFA as described in 3.3.1. To make IBV plaques visible, they were stained according to the im-munofluorescence protocol (see 3.3.2).

54 3. Methods

3.3 Lipid rafts

3.3.1 Fixation of cells

The cell layer was washed three times with 1 ml PBS per well of a 24-well plate. To each well 200µl 3 % paraformaldehyde (PFA) were added and incubated for 20 min at room temperature.

The PFA was disposed separately and cells were washed three times as before.

3.3.2 Immunofluorescence analysis

Cells grown on cover slips in 24-well plates were fixed as described in 3.3.1. Cells infected with viruses recombinant for eGFP were mounted in Mowiol while cells infected with IBV, which is not recombinant for eGFP, were immunostained first. To this end, cells were permeabilised by treatment with 0.2 % triton-X-100 for 5 min at room temperature and washed three times with 1 ml PBS. IBV-infected cells were stained with 200µl rabbit-α-IBV-serum, diluted 1:200 in PBS, for 1 h at room temperature and subsequently washed three times with 1 ml PBS. There-after cells were incubated with 200µl of goat-α-rabbit-IgG, FITC-conjugated, diluted 1:500 in PBS, for 1 h at room temperature, washed as before and mounted in mowiol.

3.3.3 Flow cytometer analysis

For each flow cytometric sample, cells of a well of a 24-well plate were used. 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 200µl trypsin were 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 preceeding washing steps and sedimented 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 sedimented at 420 g for 7 min at 4^◦C. Cells infected with a virus recombinant for eGFP were resuspended in 400µl cold 1 % paraformaldehyde, fixed for 2 h at 4^◦C, and subsequently analysed flow cytometrically. Cells infected with IBV, which is not not recombinant for eGFP, had to be immunostained prior to the flow cytometric analysis. To this end the cell pellets were incubated for 1 h at room temperature in 200µl rabbit-α-IBV-serum, diluted 1:200 in PBS. Thereafter, cells were washed three times

3.3. Lipid rafts 55

with 1 ml PBS and pelleted at 420 g for 7 min at 4^◦C. Cells were then incubated for 1 h at room temperature in 200µl of a secondaryα-rabbit-IgG, FITC-conjugated antibody, diluted 1:500 in PBS. Following three washing steps as before, the cell pellets were resuspended in 400µl PBS each and directly analysed with a flow cytometer. As a negative control mock infected cells were used.

3.3.4 Depletion of cellular cholesterol using methyl-β-cyclodextrin (MβCD)

In a total volume of 1 ml Edulb containing 5 % FCS, 2 x 10⁵ Vero cells were seeded per well of a 24-well plate and incubated at 37^◦C o/n. The next day, the cells were washed two times with 1 ml PBS and concentrations of methyl-β-cyclodextrin ranging from 0 to 15 mM, diluted in Edulb, were added to each well in a total volume of 250µl and incubated for 30 min at 37^◦C.

To remove MβCD, cells were washed three times with 1 ml PBS.

3.3.5 Determination of cellular cholesterol

Cellular cholesterol was determined using the “Cholesterin Farb Test” (Roche). 2 x 10⁷ Vero cells were seeded in a 162 cm² cell culture flask in a total volume of 30 ml Edulb containing 5 % FCS and incubated at 37^◦C o/n. The other day, the cell layer was washed twice with 20 ml PBS. Cells were either treated with 7.5 ml Edulb or with the same volume of Edulb containing 7.5 mM MβCD and incubated for 30 min at 37^◦C on a rocking platform. Thereafter, the cells were washed three times as before 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 utilizing a Thoma counting chamber. Equal cell numbers of both samples were sedimented at 400 g for 7 min at 4^◦C. The pellet was resuspended in 1 ml PBS and sedimented at 18 000 g for 1 min at 4^◦C.

Subsequently, the pellets were resuspended in 400µl lysis buffer. Cell lysis was accomplished during a 20 min incubation at room temperature, vortexing the cells every 5 min.

The determination of cholesterol was performed as described in the manufacturer’s manual. In principle, a solution containing an ammoniumphosphate buffer, methanol, katalase and acety-laceton is mixed with the cholesterol containing sample and subsequently cholesterol-oxidase is added. The cholesterol-oxidase oxidises the cholesterol under formation of hydrogen peroxide

56 3. Methods

which oxidises methanol to formaldehyde in the presence of katalase. In the presence of am-monium ions formaldehyde forms with acetylaceton a yellow lutidin dye. The concentration of the lutidin dye is equivalent to the amount of cholesterol and can be measured photometrically at 405 nm.

3.3.6 Determination of cell viability after cholesterol depletion

Vero cells were cholesterol depleted as described in 3.3.4 and subsequently cell viability was determined by propidium iodide (PI) staining. To detach the cells from the macrotiter plate, they were incubated with 200µl trypsin for 10 min at 37^◦C, resuspended in 1 ml PBS, transferred into a 1.5 ml reaction tube and sedimented at 420 g for 7 min at 4^◦C. Cells were resuspended in 500µl PBS and 2µg/ml PI were added. After 5 min incubation at room temperature, the percentage of stained cells was determined by flow cytometry. Cells which were not stained with PI were used as a negative control.

3.3.7 Infection efficiency after cell membrane cholesterol depletion

To investigate the importance of cellular cholesterol for virus entry, cellular cholesterol was depleted as described in 3.3.4. Cells were subsequently treated with 250µl medium or infected with 250µl virus (CDV-Onderstepoort, CDV-5804P, VSV or IBV) dilutions in Edulb giving rise to MOIs of 0.1, 0.05, 0.01, 0.005 or 0.001 and incubated at 37^◦C for 1 h. Thereafter, cells were supplied with 1 ml methylcellulose. Cells infected with VSV, IBV and CDV-5804P were further processed for immunofluorescence analysis (3.3.2) or flow cytometer analysis (3.3.3) 1 dpi, cells infected with CDV-Onderstepoort 2 dpi.

3.3.8 Reverse transcriptase (RT) PCR for SLAM gene detection

Different cell lines were investigated for SLAM expression. 1 x 10⁶ cells (Vero, Vero-SLAM, MDCK II, Calu-3 or B95a) were sedimented at 420 g for 7 min at room temperature and resus-pended in RLT-buffer (Qiagen). Total cellular RNA was extracted according to the manufac-turer’s manual, using the RNeasy-kit (Qiagen). Then, mRNA was extracted using the Oligotex-kit (Qiagen). RNA was transcribed into cDNA by using random hexamers as primers (for a

3.3. Lipid rafts 57

protocol refer to C.6.1, for PCR-protocol see C.7.1). Canine or simian SLAM RNA as well as GAPDH RNA as a control for mRNA extraction, were amplified (for a master-mix protocol see C.6.2, for PCR protocol see C.7.2) using specific primers (for sequences see C.5.1). PCR products were separated on a 2 % agarose gel.

3.3.9 Infection efficiency of CDV-Onderstepoort and CDV-5804P on Vero or Vero-SLAM cells

2 x 10⁵ Vero or Vero-SLAM cells were seeded on cover slips in 1 ml Edulb containing 5 % FCS per well of a 24-well plate and incubated o/n. Thereafter, cells were infected with CDV-Onderstepoort or CDV-5804P in a volume of 250µl at an MOI of 0.1, 0.05, 0.01, 0.005 or 0.001 and incubated for 1h at 37^◦C. Subsequently, the inoculum was replaced with 1 ml fresh medium containing 5 % FCS. Cells were fixed, mounted in mowiol and analysed for eGFP expression 1 dpi.

3.3.10 Infection efficiency after virus envelope cholesterol depletion

To investigate the importance of viral membrane cholesterol, 2 x 10⁵ Vero cells in a volume of 1 ml Edulb containing 5 % FCS were seeded per well of a 24-well plate and incubated at 37^◦C o/n. The next day, the cell layer was washed three times with 1 ml PBS. Virus stocks (CDV-Onderstepoort or VSV) with a titer of 8 x 10⁴pfu/ml were treated for 30 min at 37^◦C with MβCD in concentrations ranging from 0 to 20 mM. Thereafter 250µl of the virus or an equal amount of medium was applied to the cell layer. Virus binding was allowed for 1 h at 37^◦C. Subsequently, cells were washed three times as before to remove unbound virus and the cells were provided with 1 ml methylcellulose and incubated as before. For an examination of the infection efficiency the cells were processed 1 dpi either for immunofluorescence analysis (3.3.2) or for flow cytometrical analysis (3.3.3).

58 3. Methods

3.3.11 Replenishment of viral cholesterol

To confirm that removal of viral cholesterol was responsible for the reduced viral infectivity, CDV-Onderstepoort was replenished by exogenous cholesterol after MβCD treatment. After depletion of viral cholesterol as described in 3.3.10, the virus was treated with medium or con-centrations of exogenous cholesterol raning from 50 to 500µM for 30 min at 37^◦C. Thereafter, cells were infected with these virus stocks and processed for immunofluorescence or flow cy-tometry as described before.

3.3.12 Isolation of Detergent Resistant Membranes (DRMs)

3 x 10⁷ Vero cells were seeded per 162 cm² flask in 20 ml Edulb containing 5 % FCS and in-cubated o/n. The next day, cells were infected with CDV-Onderstepoort at an MOI of 0.1 in a volume of 10 ml Edulb for 1 h at 37^◦C. Thereafter, the inoculum was replaced by 20 ml Edulb containing 5 % FCS and incubated for 24 h at 37^◦C. Subsequently, the cell layer was washed once with PBSM and incubated with 5 ml 10 mM EDTA in PBSM for approximately 1 h to de-tach the cells. The cells were counted and 3 x 10⁷cells were sedimented at 1300 rpm for 7 min at 4^◦C. The cell pellet was washed twice in cold PBSM and finally resuspended in cold 300µl MB++ buffer (if less cells could be harvested the volume MB++ buffer was adjusted) and kept on ice o/n. The next day, sedimented material was carefully resuspended, the volume was ad-justed to 500µl and incubated at 37^◦C for 5 min. The cooled sample was mixed with 500µl 90 % sucrose and placed into the bottom of a SW41-tube. 10 ml of a linear sucrose gradient (40 - 10 %) was overlaid and the gradient was centrifuged for 20 h at 35 000 rpm at 4^◦C with slow acceleration and deceleration. The gradient was fractionated into 11 samples by pipetting 1 ml fractions from the top to the bottom into 1.5 ml reaction tubes. The pellet was resuspended in 1 ml MB+ buffer. All samples were stored at -20^◦C until SDS-PAGE.

3.3.13 Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Equal volumes of the sample and reducing sample buffer (2 x) were mixed and heated for 5 min at 95^◦C. SDS-polyacrylamide gels were prepared according to (Laemmli, 1970) with a 10 %

3.3. Lipid rafts 59

acrylamid/bisacrylamid concentration and a thickness of 0.75 mm. The gel was run with a constant current of 25 mA per gel.

3.3.14 Western Blot

Proteins were transferred from the SDS-gel to a PVDF membrane. The membrane was activated with 100 % methanol for 2 - 3 seconds and washed with water for 5 min. The transfer was performed with the “semi-dry” procedure. Two anode buffers, differing in the pH, and a kathode buffer were used. Blotting was done for 1 h at a constant current of 0.8 mA per cm². Thereafter, the blot was incubated in blocking buffer for 1 h at 4^◦C. Antibodies directed against CDV-H and F were diluted 10 000-fold in blocking buffer, while antibodies directed against lamp-2 and flotillin-2 were diluted 1 000-fold in blocking buffer and incubated at 4^◦C o/n. The next day, the blot was washed three times with 30 ml PBSM-Tween for 8 min at RT before addition of the secondary antibody, either anti-rabbit-IgG, horse-radish peroxidase (HRP) conjugated, or anti-mouse-IgG, HRP conjugated, both diluted 1:1 000 in PBSM-Tween and incubated for 1 h at RT. The membrane was washed three times with PBSM-Tween as before and developed with peroxidase solution.

3.3.15 Syncytia formation after cholesterol depletion

To investigate the formation of CDV syncytia after cholesterol depletion, 2 x 10⁵ Vero cells in 1 ml Edulb containing 5 % FCS were seeded per well of a 24-well plate and incubated at 37^◦C o/n. The next day, the cells were washed once with 1 ml PBS and thereafter the cells were treated with 250µl medium or infected with 250µl virus dilutions giving rise to an MOI of 0.1 for 1 h at 37^◦C. Subsequently, virus inoculum was removed by three washing steps as before and cellular cholesterol was depleted by treatment with 7.5 mM MβCD in a volume of 250µM Edulb for 30 min at 37^◦C. As a control, cells were treated with an equal volume medium.

Thereafter MβCD was removed with three washing steps as before. To prevent replenishment of cellular cholesterol either by cholesterol uptake from the medium or by endogenous syn-thesis, cholesterol-depleted cells were provided with methylcellulose containing 5 % delipized FCS, 10µM mevastatin (Mayor et al., 1998) and 25µM mevalonolactone (mevalonate, which is commonly used (Keller and Simons, 1998) was no longer commercially available), while

60 3. Methods

control cells were provided with methylcellulose containing 5 % FCS. All cells were incubated o/n at 37^◦C. Cells were fixed 1 and 2 dpi with 3 % PFA (as described in 3.3.1) and mounted in mowiol.

The same approach was also applied to Vero cells transfected with CDV H and F using Lipo-fectamine^R as described in 3.5.11, instead of CDV infection. In this experimental setup, DNA uptake was allowed for two hours. Thereafter, cholesterol depletion was accomplished as was done with infected cells. Cells were washed three times with 1 ml PBS and subsequently mounted in mowiol.

3.3.15.1 Quantification of syncytia

Syncytia were quantified by counting nuclei involved in sycnytia using the fluorescence micro-scope, taking advantage of the eGFP expression or the DAPI staining, respecitvely. Syncytia were defined as cells with 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.

3.4 Cell polarity

3.4.1 Filter supports and transepithelial resistance

To investigate the impact of cell polarity on CDV infection, filter supports were used. A fil-ter support is schematically depicted in figure 3.1. Filfil-ter supports are available in different sizes, suitable for insertion into 24- or 6-well plates, and with different pore sizes, ranging from 0.4µm up to 3µm. For our experiments filters for 24-well plates with a pore size of 1µm were used. The filter support system allows cell lines to establish polarity and enables a separate supply of the apical and the basolateral side with medium or virus inocculum.

3.4. Cell polarity 61

Basolateral chamber

Apical chamber

Cell monolayer Filter support

Porous membrane

Figure 3.1: Schematic depiction of a filter support. Filter supports are inserted into the well of a suitable plate

Generally, 2 x 10⁵ cells in a volume of 200µl suitable medium including all required additives and standard antibiotics were seeded on the porous filter of the filter support and provided with additional 800µl of the same medium in the basolateral chamber. Cells were allowed to establish polarity which was monitored by daily determination of the transepithelial resistance

Im Dokument The role of lipid rafts in Canine Distemper Virus infection (Seite 49-169)