Reovirus Activates a Caspase-Independent Cell Death Pathway

Berger, Angela K.; Danthi, Pranav

Reovirus induces caspase-independent cell death. Our experiments using the IKK inhibitor suggested that reovirus may induce cell death even in the absence of active effector caspases. To directly test the idea that T3D can evoke cell death in a caspaseindependent manner, we assessed cell death 48 h after reovirus infection in the presence of inhibitors of caspase-8 (Z-IETD-FMK) or caspase-9 (Z-LEHD-FMK) or a broad-spectrum inhibitor of caspases [Z-VAD(OMe)-FMK]. Surprisingly, we found that T3D remained capable of inducing cell death in the presence of each of these inhibitors (Fig. 3A ). To ensure that the inhibitors were functional in L929 cells at the concentration used, we assessed their capacity to prevent effector caspase activation in T3Dinfected cells. For these experiments, caspase-3/7 activity in infected cells treated with each inhibitor was quantified 24 h after infection. We found that in comparison to mock-infected cells, there was an~10-fold increase in caspase-3/7 activity by infection with T3D in the presence of DMSO (Fig. 3B ). Inclusion of inhibitors of caspase-8 or caspase-9 or inclusion of a pan-caspase inhibitor completely blocked effector caspase activation. Thus, despite being capable of blocking the proapoptotic signaling pathway activated by reovirus, these inhibitors failed to prevent virally triggered cell death. These data indicate that reovirus T3D is capable of inducing a distinct cell death pathway in L929 cells that is independent of caspase activation.