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.

The analysis of the mode of cell death after application of the pharmacological compounds began with the verification of the presence of DNA fragmentation within the floater population as a hallmark of apoptosis in RV-infected cells. Application of z-VAD-fmk was excluded from the analysis as it is a well-characterized inhibitor of apoptosis. Figure 1C shows a clear laddering of oligonucleosomal fragments and indicates that floaters possess DNA fragmentation irrespective to the application of pharmacological compounds. Figure 1D indicates that with 72 h post-infection only at late time points of infection the activity of caspase 3 and 7 as executioner caspases was detectable at a significant level in RV-infected cells. Hence, this time point was used for studying the effect of NIM811, z-VAD-fmk and PFTµ on RV-induced apoptotic signaling pathways. Figure 1E shows conventional annexin V-enhanced green fluorescent protein (EGFP)/propidium iodide (PI) staining and indicates that in comparison to the SC, the number of PI-and annexin V-EGFP-positive cells was reduced after application of z-VAD-fmk and to a lesser extent after treatment with PFTµ, but not after addition of NIM811. To extend this observation, activity of caspase 3 and 7 was determined after application of PFTµ and z-VAD-fmk. Figure 1F depicts the reduction in active caspase 3 and 7 which as expected was profound after application of z-VAD-fmk. In comparison to the SC, PFTµ also reduces the amount of active caspase 3 and 7, but not as effective as z-VAD-fmk ( Figure 1F ). Taken together, PFTµ appears to target one of the key players during RV-induced cell death.