Role of CDV receptor usage for cholesterol independence

As SLAM, the receptor for wild-type CDV, has been reported to be expressed on some subsets of leukocytes, the epithelial cell line Vero is expected to lack SLAM. Therefore, it is assumed that CDV-Onderstepoort that replicates in Vero cells uses a receptor different from SLAM.

The results from 4.1.1 indicate that SLAM-independent infection is not affected by cholesterol depletion from the plasma membrane. It was of interest to learn whether cholesterol depletion affects SLAM-mediated CDV infection. To address this question, we used Vero cells stably expressing the canine SLAM (Vero-SLAM) and a virulent, recombinant CDV strain, 5804P.

4.1.2.1 Vero-SLAM but not Vero cells express SLAM mRNA

To confirm that Vero cells do not express SLAM, mRNA of Vero cells was isolated and tran-scribed into cDNA. Successful mRNA isolation of all cell lines was proven by GAPDH ampli-fication (figure 4.7A). Finally, SLAM mRNA was amplified using primers specific for simian SLAM. As a positive control, cDNA of the marmoset cell line B95a was used. As expected, B95a cells, but not Vero cells, expressed simian SLAM (figure 4.7B). Vice versa, using primers specific for canine SLAM, we showed that the Vero-SLAM cell line expressed canine SLAM while the canine cell line MDCK served as a negative control (figure 4.7C).

A

Figure 4.7: Expression of simian and canine SLAM mRNA in different cell lines. mRNA of Vero, Vero-SLAM, MDCK and B95a cells were isolated and transcribed into cDNA using random hexamers as primers.

Non-template control (1) or cDNA of B95a (2), Vero (3), Vero-SLAM (4) or MDCK (5) was amplified using primers specific for GAPDH (A). Non-template control (1) or cDNA of B95a (2) as well as of Vero cells (3) was amplified using primers specific for simian SLAM (B), while non-template control (1) or cDNA of Vero-SLAM (2) as well as of MDCK cells (3) was amplified using primers specific for canine SLAM (C).

Products were separated on a 2 % agarose gel.

4.1. Role of cholesterol for initiation of CDV infection 77

4.1.2.2 CDV-5804P uses almost exclusively SLAM as a receptor

To demonstrate that CDV-5804P infects Vero-SLAM cells by using SLAM as a receptor, Vero as well as Vero-SLAM cells were infected with CDV-5804P at different MOIs. For comparison, cells were also infected with CDV-Onderstepoort at different MOIs. Infection efficiencies were determined by flow cytometry and by fluorescence microscopy. Figure 4.8 demonstrates that CDV-5804P infection of Vero cells at an MOI of 0.1 is limited to single cells, while infection of Vero-SLAM at the same MOI included almost 100 % of the cells and resulted in massive syncytium formation and even cell lysis. CDV-Onderstepoort infection of Vero cells at an MOI of 0.1 was much more efficient compared to infection by 5804P, confirming that CDV-Onderstepoort, but not CDV-5804P, can use a receptor different from SLAM to infect Vero cells with high efficiency (figure 4.8). As for CDV-5804P, CDV-Onderstepoort infection of Vero-SLAM cells was much more efficient than Vero cell infection and also resulted in much stronger syncytium formation. These data show that CDV-5804P infects Vero-SLAM cells almost exclusively by using SLAM as receptor, while CDV-Onderstepoort can use SLAM as well as an additional receptor(s) that has not yet been identified.

The data obtained by flow cytometry did not match the results seen by fluorescence microscopy.

Infection rates for Vero-SLAM cells were much lower compared to data obtained by fluores-cence microscopy (data not shown). This is thought to be due to the massive syncytium forma-tion seen on Vero-SLAM cells, resulting in cell detachment and destrucforma-tion, thereby interefering with flow cytometrical measurement. Therefore, for all further experiments data were exclu-sively obtained by fluorescence microscopy.

78 4. Results

Vero Vero-SLAM

CDV-5804P - CDV- Onderste-poort

Figure 4.8: CDV-5804P and CDV-Onderstepoort infection of Vero and SLAM cells. Vero or Vero-SLAM cells were infected with CDV-5804P or CDV-Onderstepoort at an MOI of 0.1 and prepared for fluorescence microscopy 1 dpi. Pictures were taken at a 100 x magnification. Results are representative for three independent experiments.

4.1.2.3 CDV-5804P infects Vero-SLAM cells independently of cell membrane cholesterol

To investigate if cell membrane cholesterol is required for infection by CDV when SLAM is used as a receptor, Vero-SLAM cells were pretreated with increasing MβCD concentrations and subsequently infected by CDV-5804P and, as controls, by IBV, VSV or CDV-Onderstepoort, respectively. Infection efficiencies for all viruses were determined by fluorescence microscopy 1 dpi. Figure 4.9 A demonstrates that CDV-5804P infects Vero-SLAM cells independently of cell membrane cholesterol and that this effect was also independent of the MOIs used (fig-ure 4.9 B). IBV, VSV and CDV-Onderstepoort behaved in the same way on Vero-SLAM cells as on Vero cells (data not shown). Therefore, we conclude that infection by CDV is not af-fected by cholesterol depletion from the cell membrane, irrespectively of the receptor used for infection. The implications of these results for the localisation of the receptor are discussed in 5.1.

4.1. Role of cholesterol for initiation of CDV infection 79

MOI 0.1 0mM M CDb 7.5mM M CDb

Mock

0mM

3mM

7.5mM

15mM

0.001

0.005

0.01

0.05 Mock

M CDb MOI

A B

Figure 4.9:Infection of cholesterol-depleted Vero-SLAM cells by CDV-5804P. (A) Vero-SLAM cells were treated with increasing MβCD concentrations for 30 min at 37^◦C and subsequently infected by CDV-5804P at an MOI of 0.1. Samples were prepared for fluorescence microscopy 1 dpi and pictures were taken with 100 x magnification. Results are representative of three independent experiments. (B) Vero-SLAM cells were treated with medium or 7.5 mM MβCD for 30 min at 37^◦C and subsequently infected by CDV-5804P at different MOIs. Samples were prepared for immunofluorescence 1 dpi and pictures were taken with 100 x magnification. Results are representative of three independent experiments.

80 4. Results