Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection

Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K.; Liebert, Uwe G.

Immunofluorescence analysis of RV-and mock-infected cells was applied to validate CytC localization during RV infection. MitoTracker Red CMXRos (Thermo Fisher Scientific, Braunschweig, Gemany) was used as a mitochondrial counterstain. Figure 4A highlights the strict mitochondrial localization of CytC in mock-infected Vero cells. (A) Immunofluorescence analysis of RV-and mock-infected Vero cells at 3 dpi with anti-CytC antibody (shown in green). As a counterstain, mitochondria were labelled with MitoTracker Red CMXRos (shown in red) before fixation. Nuclear DNA is shown in blue, yellow colour indicates overlap areas. As a positive control, overnight incubation with 0.003% H2O2 was applied; (B) Selective permeabilization of the plasma membrane with digitonin was tested for alpha-tubulin (shown in red) as a representative cytoplasmic protein; (C) Selective permeabilization with digitonin at 25 μg/mL was applied to mock-and RV-infected Vero cells. Immunofluorescence was performed with anti-CytC antibody (shown in red). Release of CytC was induced through overnight incubation with 0.003% H2O2. (B), (C) As a control, immunofluorescence analysis of mock-infected Vero cells was performed after methanol (Met-OH) permeabilization. Asterisks highlight representative cells with localization of CytC outside of mitochondria. Scale bar, 10 μm. Release of the mitochondrial death-effector protein AIF. Vero cells overexpressing AIF-TagRFP were applied at 2 dpi (A) for determination of RV-induced translocation of AIF-TagRFP (shown in red) from mitochondria to the nucleus. Nuclear DNA is shown in blue, the viral E1 protein is shown in green. Asterisks (mock and Aa) indicate physiological localization of AIF to mitochondria. Arrows (Ab,c and d) highlight cells with nuclear association of AIF; (B) 30 µg of mitochondrial fractions obtained at 3 dpi from mock-and RV-infected Vero cells were subjected to Western blot analysis with anti-AIF antibody. As a loading control, anti-VDAC antibody was applied. Scale bar, 10 µm.