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.

p53-and cyclophilin-regulated pathways are generally altered in cancer cells, hence experiments were mainly performed with Vero, a standard and non-tumor-derived cell line for RV cultivation. Additionally, CPE by RV is very profound in this cell line [18] . Several RV-associated cell culture aberrations were characterized in this cell line, including the analysis of the p53 dependency [5] . Vero cells are negative for the type I interferon response [44] , which allows the separation of RV-induced effects on mitochondria-based pathways from effects that are triggered through generation of type I interferons [45] . Vero and A549 cells were maintained in DMEM, supplemented with fetal bovine serum (10%) and antibiotics at 37 ¥ C in a humidified incubator with 5% CO 2 atmosphere. Medium for A549 cells was supplemented with 1 mM HEPES. After overnight cultivation, cells were mock-and RV-infected and treated with pharmacological compounds or vehicle (solvent control) only. Medium was removed and effectors were added in fresh medium at indicated time points. Cells were incubated in the presence of the respective effector until sample processing. The laboratory-adapted F-Therien strain was applied to Vero cells at an multiplicity of infection (MOI) of 5 unless otherwise indicated. Besides the Therien strain, the vaccine strain HPV77 and the clinical isolate Wb-12, a kind gift of B. Weißbrich (University of Wuerzburg, Wuerzburg, Germany), were used. The Wb-12 strain was passaged seven times on Vero cells after its initial isolation. An MOI of 5 was chosen to ensure an optimal infection rate resulting in a high level of CPE induction. The application dose of the pharmacological compounds was assessed by classical trypan blue exclusion test and was within the published range.