Phenotyping of wild type and integrin deficient mouse fibroblats

expression features. Integrins are expressed as heterodimeric receptors on the cell surface (2.7.1). Thus, the loss of the αv integrin subunit results in the lack of all five αv integrin receptor combinations (αvβ1, αvβ3, αvβ5, αvβ6, αvβ8; see also 2.7.4). In contrast, on β3-deficient MEFs only the expression of integrin αvβ3 is affected. Since the alternative β3 integrin, i.e. αIIbβ3, is exclusively expressed on thrombocytes it is not relevant in this context.

Two additional mouse fibroblast (in the following: MF) cell lines were made available by R.

Faessler (A.1.7.4): mouse kidney fibroblasts MKF-ITGB1(-/-) and their parental cell line ITGB1(flox/flox). Thus, the latter was used as a wild type control for the MKF-ITGB1(-/-). The term ‘flox’ refers to the genetical marking of a gene sequence by LoxP sites

in order to be able to delete the corresponding gene fragment. Both cell lines were immortal-ised by the SV40 large T antigen (R. Faessler, Max Planck Institute, Martinsried, Germany;

pers. comm.) and differed from the MEFs described above in their morphology and the proliferation rate. The absence of the integrin subunit β1 makes the expression of 12 hetero-dimeric integrin combinations, i.e. integrin αvβ1 and integrins α(1-11)β1, impossible.

4.3.4.1 Characterisation of the MF cell lines by immuno-fluorescence staining In order to verify the αv, β1 and β3 integrin deficiency of the established cell lines and to examine the distribution of these integrins assembled in focal contacts on the cell surface of MFs immuno-fluorescence was accomplished using mouse-specific antibodies directed against the αv (mab clone RMV-7), β1 (mab HMβ1-1) or the β3 (mab HM beta 3.1) subunit.

At sub-confluency, 24 to 48 hours after seeding onto glass cover slips, cells were fixed and stained before integrin distribution and nuclear staining were visualised by confocal laser scanning microscopy. Sub-confluency was important to picture the focal contact rich cell protrusions and cell-to-cell-contacts. As seen in Figure 19 imaging showed that αv and β3 integrins assembled in a typical focal contact manner, i.e. isolated bar-shaped dots, localised on the cell surface. Double fluorescence staining revealed that αv integrins were concentrated in the cell periphery whereas β3 integrins were also found in the inner cell radius. Most noticeably, the distribution of β1 integrins showed a different picture. They presented a more elongated assembly across the cell surface similar to those of stress fibres with which they are connected. The same structures were seen in all but the β1-deficient MKFs, so that the antibody reaction was regarded to be specific. The images in Figure 19 show that both αv and β3 integrins are absent from the surface of MEF-ITGAV(-/-) cells derived from the homozy-gous αv-mutant embryos. Apart from the absence of detectable β3 integrins on the surface of MEF-ITGB3(-/-) cells there were no changes in the expression pattern regarding αv integrins.

The expression of β1 integrins of both deficient cell lines was not altered. The MKF- ITGB1(-/-) cells lacked β1 integrins but showed the expected distribution of αv and β3 integrins. All these integrins were found on the surface of the two wild type cell lines, wild type MEFs and MKF-ITBG1(flox/flox).

Figure 19. Confocal microscopy images of integrin expression and distribution of the MFs used in this study. Antibodies specific for the integrin subunits αv, β1 or β3 and the combi-nation of αv (green) and β3 (red) integrin specific antibodies were used to picture integrin expression. Panels show images for each combination of cell line × antibody. Nuclear stain-ing was realised by the nucleic acid stain DAPI. Merged colours of the double stainstain-ing indi-cate co-localisation of integrins (yellow). Scale bars of 100 µl (light microscopy images) and 20 µm (fluorescence images) are displayed.

In addition to the MF cell lines, the integrin αvβ3-specific antibody LM609 raised against the human heterodimeric integrin αvβ3 was used to picture the integrin distribution on the surface

of Vero cells. Images in Figure 20 show that Vero cells have a comparably high expression density of integrin αvβ3 as reported before [99]. Application of this antibody to the MFs in combination with secondary anti-mouse IgG antibodies failed. Binding of the monoclonal antibody LM609 to the mouse cells was non-specific.

Figure 20. Immuno-fluorescence staining of integrin αvβ3 on the surface of Vero cells.

Monoclonal integrin αvβ3-specific antibody LM609 and a secondary anti-mouse Alexa Fluor®546 antibody were used. (A) Cell clustering, (B) two cells. Scale bars of 20 µm are displayed.

4.3.4.2 Characterisation of the MF cell lines by flow cytometry analysis

Flow cytometry analysis was used to estimate the integrin expression levels on live cell surfaces of the wild type and integrin deficient MFs. The fluorescence profiles were measured using the FACSalibur flow cytometer after incubation with anti-mouse integrin specific antibodies targeting the αv subunit (mab RMV-7), the β3 subunit (mab HM beta 3.1 and 2C9.G2) or the β1 subunit (mab HMβ1-1). Control cells incubated with isotype antibodies showed similar fluorescence intensities to untreated cell controls. 2 × 10⁵ cells were stained and a minimum of 10.000 cells were counted at a time. Cells were gated upon morphological characteristics whereby dead cells were excluded from FACS analysis. In general, the results of the flow cytometry analysis were consistent with the PCR and confocal laser scanning microscopy results described in 4.3.3 and 4.3.4.1, respectively. After subtracting the back-ground fluorescence of the cell control no β3 integrin positive cells were found in the scatter plots of MEF-ITGB3(-/-) whereas all cells were found to be positive for αv and β1 integrins.

Expression of αv and β3 integrins on MEF-ITGAV(-/-) was below the limit of detection.

Fluorescence signals did not exceed the baseline when MKF-ITGB1(-/-) cells were incubated with the β1-specific antibody, indicating the absence of β1 integrins, whereas the expression of αv and β3 integrins was not affected. Figure 21 shows the integrin expression profiles

represented by FACS histograms. The untreated cell controls were adjusted to the same fluorescence intensity level in order to compare fluorescence levels of the different cell lines.

Interestingly, no significant differences between fluorescence signals were seen. These findings suggest that the β1 integrins did not show any evidence of up-regulation in expres-sion in compensation for the absence of β3 integrins or vice versa. It was concluded that the integrin expression levels of the MEF cell lines are comparable. The expression levels of αv and β3 integrins on MKF-ITGB1(-/-) and MKF-ITGB1(flox/flox) were reduced in compari-son to the established cell lines.

Figure 21. Fluorescence profiles from flow cytometry analysis. Cells were incubated with integrin specific antibodies or left untreated (cell controls, isotype controls not shown here).

Panels represent FACS histogram for each combination of cell line × antibody. Monoclonal antibodies RMV-7, HMβ1-1 and HM beta 3.1 were used for detection. Abscissa - fluores-cence intensity (log scale); ordinate - number of cells counted in each column of the histo-gram.

4.3.4.3 Characterisation of the MEFs by mouse adenovirus type 1

Deficiency of a cell line with respect to a particular receptor should result in a resistance or partial resistance to infection by a control virus that uses the same receptor. However, the difficulty is that most virus species that have been described to use integrins for viral entry can use alternative receptors in the absence of integrins or do not use exclusively integrins, respectively. Some human adenoviruses use integrins as co-receptors for entry after binding to the coxsackie-adenovirus-receptor CAR [351, 508]. Human adenovirus type 5 which is dependent on αv integrins as co-receptors was kindly provided by Kathrin Zimmermann, University of Greifswald, Germany. However, first experiments showed that virus replication in the mouse cell lines ceased, so that it could not be used as a control virus for the MEFs.

Since the murine adenovirus type 1 (MAV-1) has been shown recently to also be dependent

on αv integrins for entry [389] it was used instead. MAV-1 replicated in the MEFs and showed a strong cytopathic effect.

Wild type MEFs, MEF-ITGB3(-/-), MEF-ITGB3(-/-)rescue and MEF-ITGAV(-/-) were seeded onto 12-well culture dishes 24 hours prior to infection. The protocol for infection experiments given in Materials and Methods (3.7.1) was generally followed. More precisely, 1 × 10⁵ cells per well were pre-cooled before inoculated with virus at an MOI of 0.5 PFU/cell.

Infection was allowed for 90 minutes at 4 °C. To determine yields of virus bound to the cell surface a first set of cells was harvested after extensive washing with PBS. The second set was washed before maintenance medium was added to the wells and incubation proceeded for 2 days at 37 °C. Virus DNA was isolated from the supernatant and from the TRIzol samples from the first set before being quantified by qPCR to calculate ct-values.

The Kruskal-Wallis test was used for statistical analysis of the real-time PCR read-out.

Comparison of the rank means was accomplished by the Tukey-Kramer’s HSD test. Figure 22-A shows that binding to MEF-ITGAV(-/-) was significantly reduced compared to wild type MEFs as had been described by Raman et al. [389]. A more prominent, highly significant effect had the loss of the integrin β3 subunit on virus binding which led to lower yields in MAV-1 ct-values by a factor of 10^-3. Most noticeably, when the integrin β3 subunit was rescued as seen in MEF-ITGB3(-/-)rescue (establishment of these cells see 4.4.3.3), binding efficiency of MAV-1 was comparable with wild type cells. After incubation of cells with MAV-1 for 48 hours the highly significant difference between wild type and MEF-ITGB3(-/-) disappeared (Figure 22-B). Here, the absence of the αv subunit had an enhancing effect on MAV-1 replication which was completely unexpected. Levels of virus production were similar to those seen in β3-rescue MEFs. These findings clearly demonstrate that β3 integrins are involved in (i) MAV-1 binding, and (ii) replication, both effects being independent from each other. Presumably this is due to an improved uptake of virus particle into MEF- ITGB3(-/-)rescue cells. The lack of αv integrins led to reduced yields of bound virus but obviously this did not influence virus production negatively, instead it enhanced virus’

replication compared to wild type cells. To address this issue, additional experiments would have been necessary to clarify the role of αv integrins in terms of MAV-1 replication. This, however, was beyond the scope of this study. Nevertheless, results clearly demonstrate that the presence or absence of both, αv and β3 integrins, led to different binding and replication efficiencies of MAV-1.

8

Figure 22. Binding of MAV-1 to and replication in MEFs. (A) Binding and (B) replication of virus after 48 hrs p.i., both quantities expressed in ct-values. Testing: (A) Kruskall-Wallis test, n =3, χ² = 9.53, p < 0.03, (B) Kruskal-Wallis test, n = 3, χ² = 8.95, p < 0.03, multiple comparisons: Tukey-Kramer HSD test, (n.s.) p > 0.05, (*) 0.01 < p ≤ 0.05, (**) 0.001 < p ≤ 0.01, (***) p ≤ 0.001.

4.4 Cell infection studies

The establishment of cell lines that are deficient for specific integrin subunits had been outlined in the previous chapter. In addition to PCR analysis MFs were characterised as to their expression features concerning the three mentioned integrin subunits. In the following the results of several infection experiments are described that were conducted in order to identify the specific role of integrins in WNV entry. Other proteins were also considered as to their involvement in virus binding and internalisation.