Role of viral cholesterol for CDV-Onderstepoort infection

Since viruses may depend on the cholesterol content within the virus envelope membrane, the dependence of CDV-Onderstepoort on envelope cholesterol for entry into Vero cells was exam-ined.

4.1.3.1 Infection efficiency of cholesterol-depleted CDV-Onderstepoort is decreased

Canine distempervirions were cholesterol-depleted by treatment with increasing MβCD con-centrations and subsequently used to infect Vero cells. As a negative control,Vesicular stomati-tisvirions were equally treated and used for infection. Infection efficiencies were determined by flow cytometry and fluorescence microscopy 1 dpi. Figure 4.10 shows that CDV infection is reduced in a concentration-dependent manner, dropping to approximately 30 % of the infection reached by untreated virions when pretreated with 5 mM MβCD. In contrast, VSV infection was even increased by treatment with MβCD concentrations up to 5 mM, but decreased by treatment with higher concentrations. Since CDV-Onderstepoort infection was already reduced by treatment with lower concentrations, which was also seen by fluorescence microscopy analy-sis (figure 4.11), CDV appeared to require envelope cholesterol for efficient infection.

0

Figure 4.10: Effect of cholesterol depletion of the CDV-Onderstepoort or VSV envelope on infectivity.

CDV or VSV were treated with increasing MβCD concentrations for 30 min at 37^◦C and subsequently used for infection of Vero cells. Infection efficiencies were determined 1 dpi by flow cytometry. Infection rates for untreated virions were set to 100 %. Results are mean values of three independent experiments with standard deviation (standard deviations for some CDV values are not visible as they are smaller than the data point).

4.1. Role of cholesterol for initiation of CDV infection 81

Mock 0mM

3mM 5mM 10mM

1mM

Mock 0mM 1mM

3mM 5mM 10mM

CDV

VSV

Figure 4.11: Effect of cholesterol depletion of the CDV-Onderstepoort or VSV envelope on infectivity.

CDV or VSV were treated with increasing MβCD concentrations for 30 min at 37^◦C and subsequently used for infection of Vero cells. Infection efficiencies were determined 1 dpi by fluorescence microscopy and pictures were taken with 100 x magnification. Results are representative of three independent experiments.

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4.1.3.2 Replenishment of virus envelope cholesterol restores CDV-Onderstepoort infec-tivity

To confirm that cholesterol reduction was responsible for the decreased infectivity observed with CDV, cholesterol was depleted as described above and then replenished by exogenous sources prior to infection. Supplementation with only 50µM cholesterol increased infectivity when compared to samples which were not replenished and 100µM cholesterol even restored infectivity to approximately 80 %, as determined by flow cytometry (figure 4.12). Addition of cholesterol concentrations higher than 100µM were less efficient. The same results were obtained when infectivity was determined by fluorescence microscopy (figure 4.13). These data indicate that CDV requires cholesterol in its envelope membrane for efficient entry.

0 20 40 60 80 100

0 50 100 200 500

Cholesterol [µM]

Infectedcells[%]

Figure 4.12:Replenishment of envelope cholesterol prior to infection by CDV.Canine distempervirions were cholesterol depleted by treatment with 5 mM MβCD for 30 min at 37^◦C and immediately replenished by treatment with increasing concentrations of exogenous cholesterol for 30 min at 37^◦C and subsequently used for infection of Vero cells. Infectivities were determined by flow cytometry 1 dpi. Infection rates for untreated virions were set to 100 %. Results are mean values of three independent experiments with standard deviation.

4.1. Role of cholesterol for initiation of CDV infection 83

Untreated

Mock CDV

5mM M CD treated

b

0µM cholesterol 50µM cholesterol

100µM choelsterol 200µM cholesterol

Figure 4.13:Replenishment of envelope cholesterol prior to infection with CDV.Canine distempervirions were cholesterol depleted by treatment with 5 mM MβCD for 30 min at 37^◦C, immediately replenished by treatment with increasing concentrations of exogenous cholesterol for 30 min at 37^◦C. Control virions were only treated with medium, and subsequently used for infection of Vero cells. Samples were prepared for fluorescence microscopy 1 dpi and pictures were taken with 100 x magnification. Results are representative of three independent experiments.

84 4. Results

4.2 Role of cholesterol for late steps in the CDV replication cycle

For some viruses it has been described that viral proteins are located within DRMs and it has been concluded that lipid rafts may act as platforms for assembly and budding for these viruses.

The fact that CDV envelope cholesterol is required for infection of CDV implies that cellular cholesterol is incorporated into virus particles and thus has to be present during the assembly and budding process. Therefore, cholesterol may play a functional role during late steps of the CDV replication cycle.

4.2.1 CDV H and F but not M partially associate with DRMs in infected Vero cells

The dependence of CDV infection on cholesterol within the viral envelope suggests that the CDV envelope proteins and/or the matrix protein are located within lipid rafts. To obtain exper-imental evidence, DRMs were isolated from CDV infected Vero cells by lysis with 1 % Brij98 and linear sucrose gradient centrifugation. As shown in figure 4.14, CDV H and F proteins par-titioned into low density fractions similar to flotillin-2, which is known to be a DRM-resident protein (Baumann et al., 2000; Laliberte et al., 2006). In contrast, the distribution of the M protein in the gradient fractions rather resembled the distribution of lamp-2, a protein known not to partition into DRMs (Karacsonyi et al., 2005) (figure 4.14). Association of the F protein with DRMs appears to be stronger than that of the H protein.

4.2.2 Role of cellular cholesterol for CDV-induced syncytium formation

Syncytium formation is one event during the late phase of CDV replication. Syncytium forma-tion requires the binding of one cell to another via the CDV attachment protein H, the close interaction of the CDV envelope proteins H and F to trigger the fusion process and the CDV F protein, which finally mediates the fusion. During all these steps, cholesterol may play a func-tional role. Therefore, the role of cholesterol during CDV-induced syncytium formation was analysed.

4.2. Role of cholesterol for late steps in the CDV replication cycle 85

Flotillin-2 CDV F1 CDV H Lamp-2 1 2 3 4 5 6 7 8 9 10 11 P

CDV M

Figure 4.14:Distribution of CDV envelope proteins in DRMs. CDV infected Vero cells were lysed in 1 % Brij98 and subjected to linear sucrose gradient centrifugation. 11 fractions of the same volume and the pellet solubilised in the same volume were resolved by SDS-PAGE. The CDV proteins H, F, and M as well as the cellular proteins lamp-2 and flotillin-2 were detected by Western Blot analysis.

4.2.2.1 Cholesterol starvation of Vero cells for two days does not affect cell viability

To investigate whether cholesterol has a functional role during late steps of the CDV replication cycle, Vero cells were grown under cholesterol starving conditions. Cells were cholesterol-depleted by treatment with 7.5 mM MβCD for 30 min. To prevent cholesterol replenishment af-ter removal of MβCD, endogenous cholesaf-terol synthesis was inhibited by the drug mevastatin, which blocks HMG-CoA-reductase. The maintenance of other metabolic pathways downstream of HMG-CoA-reductase block was allowed by addition of low concentrations of mevalonolac-tone. Cholesterol replenishment by uptake from the medium was prevented by addition of delipized FCS. Cells grown under such conditions for one or two days were monitored for cell viability. Microscopic examination of the cells two days after the start of cholesterol starvation revealed that the growth rate might be slightly diminished but otherwise there was no increase in the number of morphologically altered or detached cells visible (figure 4.15). Additionally, PI incorporation quantified flow cytometrically did not show any increase in the number of dead cell (figure 4.16).

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Figure 4.15: Microscopic picture of Vero cells grown under cholesterol starving conditions. Vero cells were cholesterol depleted by treatment with 7.5 mM MβCD for 30 min at 37^◦C followed by treatment with 10µM mevastatin, 25µM mevalonolactone and 5 % delipized FCS for one or two days. Control cells were treated with medium and grown in medium containing 5 % normal FCS. Cell morphology and viability was monitored microscopically and pictures were taken with 100 x magnification. Results are representative of three independent experiments.

Figure 4.16: Vero cell viability after growth under cholesterol starving conditions. Vero cells were in-cubated with 7.5 mM MβCD for 30 min at 37^◦C followed by treatment with 10µM mevastatin, 25µM mevalonolactone and 5 % delipized FCS for one or two days. Control cells were treated with medium and grown in medium containing 5 % normal FCS. Subsequently, cell viability by means of PI incorporation was measured by flow cytometry. Results are mean values of three independent, quadrouble experiments with standard deviation.

4.2. Role of cholesterol for late steps in the CDV replication cycle 87

4.2.2.2 Cholesterol starvation of cells during CDV infection decreases syncytium forma-tion

To analyse the involvement of cellular cholesterol in the syncytium formation after CDV in-fection, Vero cells were cholesterol-depleted one hour post infection and subsequently kept under cholesterol starving conditions for one or two days. To demonstrate that virus propa-gation in such metabolically restricted cells was still possible, control cells were infected by VSV. Syncytium formation in CDV-infected cells or plaque formation in VSV infected cells was monitored one and two dpi. Infected cells as well as syncytia were easily detectable by eGFP expression of the recombinant viruses by fluorescence microscopy. Control cells infected by CDV-Onderstepoort grown under normal conditions developed one dpi many small syncytia consisting of just a few fused cells as well as some large syncytia. In contrast, in cholesterol starved-cells onedpi only a few small syncytia had developed, while no large syncytia were ob-served (data not shown). Two dpi the difference in the microscopic appearance of cells grown under normal conditions compared to those grown under cholesterol starving conditions was even more pronounced. When grown under normal conditions, almost all cells were infected and lots of large syncytia were detected whereas almost no large syncytia were detected in cells grown under cholesterol starving conditions (figure 4.17). Plaque formation in VSV-infected cells was not affected when cells, grown under cholesterol starving conditions, were analysed by fluorescence microscopy. One dpi, single plaques with normal variation in size and shape were observed in control as well as in treated cells (figure 4.17), while two dpi almost 100 % of the cells were infected, with many cells having already detached from the cover slip. Here, too was no difference detectable between normally grown cells and those grown under cholesterol starving conditions (data not shown).

88 4. Results

Untreated

Cholesterol depleted

100x GFP

200x GFP

200x Light CDV

Untreated

Cholesterol depleted VSV

Figure 4.17:Effect of cholesterol depletion on syncytium or plaque formation, respectively in CDV or VSV infected cells. Vero cells were infected with CDV-Onderstepoort or VSV (both recombinant for eGFP) at an MOI of 0.1 for 1 h at 37^◦C and subsequently cholesterol-depleted by treatment with 7.5 mM MβCD for 30 min at 37^◦C or medium treated as a control. To prevent cholesterol replenishment, cells were treated with mevastatin, mevalonolactone and delipized FCS. Control cells were treated with medium containing normal FCS. VSV samples were prepared for fluorescence microscopy 1 dpi, CDV samples 2 dpi and pictures were taken with 100 x or 200 x magnification. Results are representative for three independent experiments.

4.2. Role of cholesterol for late steps in the CDV replication cycle 89

4.2.2.3 Quantification of syncytia

To quantify the effect of cholesterol starvation during CDV infection on syncytium formation the number and the size of syncytia were determined for samples fixed one dpi using a counting grid. Quantification of syncytia two dpi was not possible as some syncytia had already detached from the cover slip. Syncytia were defined as cells containing more than three nuclei and the size of syncytia was defined by the number of nuclei involved in one syncytium. Figure 4.18 shows that the number of syncytia in cells grown under cholesterol starving conditions was reduced by 77 % and the size by 68 %. Altogether, cholesterol starvation during the course of CDV infection reduced the number of nuclei involved in syncytia by 93 %.

0 5 10 15 20

syncytia per field nuclei per syncytium

Number of nuclei per syncytium or syncytia per field Medium treated Cholesterol depleted

Figure 4.18: Quantification of CDV-induced syncytium formation in cells grown under normal condi-tions compared to cells grown under cholesterol starving condicondi-tions. Vero cells were infected with CDV-Onderstepoort at an MOI of 0.1 for 1 h at 37^◦C and subsequently cholesterol depleted by treatment with 7.5 mM MβCD for 30 min at 37^◦C or medium treated as a control. To prevent cholesterol replenishment, cells were treated with mevastatin, mevalonolactone and delipized FCS. Control cells were treated with medium containing normal FCS. Samples were fixed 1 dpi and syncytia quantified by counting the nuclei or syncytia, respectively under the fluorescence microscope. Results are mean values of three independent experiments with standard deviation.

90 4. Results

4.2.2.4 Cellular cholesterol starvation decreases syncytium formation in CDV H and F transfected cells

The dependence on cholesterol for syncytium formation in CDV-infected cells was confirmed with Vero cells transfected with plasmids encoding the CDV H and F genes. Figure 4.19 demon-strates that syncytium formation in transfected cells is inhibited one day post transfection as well as two days post transfection.

Untreated

Cholesterol depleted

1 day post transfection 2 days post transfection 2 days post mock transfection

Figure 4.19:Effect of choletsterol depletion on syncytium formation in Vero cells transfected with CDV H and F. Vero cells were transfected with CDV H and F or mock transfected. Two hours post transfection, cells were cholesterol depleted by treatment with 7.5 mM MβCD for 30 min at 37^◦C and subsequently treated with 10µM mevastatin, 25µM mevalonolactone and 5 % delipized FCS. Control cells were first treated with medium and subsequently treated with medium containing 5 % normal FCS. Cells were prepared for microscopy one or two days post transfection, respectively. Pictures were taken with 200 x magnification and are representative of three independent experiments.

4.3 Entry of CDV into polar cells

Beside the functional role of cholesterol for CDV replication, we were also interested in the impact of cell polarity on CDV infection. It is known that CDV is transmitted by aerosoles, thus polarised epithelial cells of the respiratory tract are candidates as primary target cells for CDV. Late in infection, CDV is shed by the epithelial cells lining the respiratory tract as well

4.3. Entry of CDV into polar cells 91

as the urinary tract. The possible consequences and restrictions associated with the cell polarity for CDV infection and release have not yet been investigated for this paramyxovirus.

4.3.1 Entry of CDV into several polar cell lines is inefficient

Therefore, we wanted to investigate whether the entry of CDV into polar cell lines is restricted to either the apical or the basolateral side. Several cell lines known to develop polarity in cell culture were grown to confluency on filter supports. Establishment of polarity was verified by measuring the transepithelial resistance (TER) which normally peaked one to two days post seeding, thereafter declining and then remaining at a constant level for several days. The cells were infected with CDV-Onderstepoort one day post seeding either from the apical or basolat-eral side, respectively. The cell line Vero C1008 did not establish a measurable TER and these cells were massively infected with CDV-Onderstpoort from both sides. All other cell lines tested established a measurable TER but were infected with CDV with only low efficiency, irrespec-tively of the side of inoculum application (figure 4.20). Interestingly, all of these cell lines were infected quite efficiently, when infected during the seeding of the cells, i.e. before cell polar-ity was established. Nevertheless, infection from the apical side was generally slightly more efficient than that from the basolateral side.

4.3.2 Entry of CDV into MDCK II after cellular depletion of bivalent ions is inefficient

It has been shown previously by Marozin et al. (2004) that Herpes simplex virus 1, which in-fects polarised epithelial cells from the basolateral membrane, is able to infect cells from the apical side after loss of cell polarity by depletion of bivalent ions with EGTA. A similar ex-periment was performed with CDV. As a positive control for this exex-perimental setup, MDCK II cells treated with PBS, PBSM or EGTA in PBSM were infected with VSV. A first experiment showed that infection of VSV from the apical side is drastically enhanced upon pretreatment with PBSM compared to treatment with PBS, probably due to the fact that PBSM does not con-tain bivalent ions. The results showed that PBSM treatment depolarised MDCK II cells to allow apical infection by VSV. Measurement of the TER confirmed that PBSM was sufficient to

depo-92 4. Results

Figure 4.20:CDV-Ondestepoort infection of different cell lines. (A) Vero, MDCK I, MDCK II or Calu-3 cells were seeded on filter supports and grown to confluency for one day. Subsequently, cells were infected from the apical or the basolateral side, respectively with 1 x 10⁶pfu/ml and prepared for fluorescence mi-croscopy 2 dpi. Pictures were taken with a 100 x magnification. (B) Vero, MDCK I, MDCK II or Calu-3 cells were infected with 1 x 10⁶pfu/ml while seeding on cover slips. Cells were prepared for fluorescence microscopy 2 dpi and pictures were taken with 100 x magnification.

larise the cells. Therefore, MDCK II cells were grown to confluency on filter supports. One day post seeding the polarisation of the cells was confirmed by measuring the TER, which reached values between 900 - 1200 Ohm x cm². Prior to infection with CDV-Onderstepoort or the con-trol VSV, cells were treated with PBSM or with PBS (which contains bivalent ions) at 37^◦C for 30 min. The TER in PBSM-treated cells decreased to values between 70 - 100 Ohm x cm², while the TER for PBS-treated samples was nearly unaffeted. Figure 4.21 shows that VSV infection from the apical side was much more efficient upon PBSM treatment compared to MDCK II cells treated with PBS prior to infection, indicating that the experimental setup allows efficient VSV infection from the apical side. In comparison to the results obtained for VSV, CDV infection of MDCK II cells from the apical side was not enhanced upon treatment with PBSM prior to

4.3. Entry of CDV into polar cells 93

infection when compared with cells treated with PBS (figure 4.21). Thus, infection of polarised MDCK II cells by CDV is inefficient, both from the apical as well as from the basolateral side.

PBS PBSM

VSV

CDV

Figure 4.21: Apical infection of MDCK II cells with 6 x 10⁸pfu/ml VSV or with 3.4 x 10⁵pfu/ml CDV-Onderstepoort after depolarisation by depletion of bivalent ions. MDCK II cells were seeded on filter sup-ports and grown to confluency for two day. Prior to infection, cells were treated with PBSM or PBS as a control for 30 min at 37^◦C. VSV infected cells were prepared for fluorescence microscopy 7 hpi, CDV infected cells 2 dpi. Pictures were taken with 100 x magnification.

4.3.3 Apical entry of CDV in primary canine epithelial cells of the respi-ratory tract

To investigate the polar entry of CDV with an in vitro-system which most closely resembles the natural infection, primary epithelial cells of the trachea and upper bronchi of canine lungs were isolated and grown in culture. The first passage was grown on filter supports and infected with CDV and with VSV as a control either from the apical or from the basolateral side two days post seeding. Cells had established a TER of 130 - 170 Ohm x cm² at day one, which only increased slightly on day two when it reached values between 170 - 230 Ohm x cm². Figure 4.22 shows that VSV infection was more efficient from the basolateral side, indicating that cells

94 4. Results

successfully established polarity, as was also obvious from the TER. In contrast, CDV infection

successfully established polarity, as was also obvious from the TER. In contrast, CDV infection

Im Dokument The role of lipid rafts in Canine Distemper Virus infection (Seite 80-100)