Reovirus Activates a Caspase-Independent Cell Death Pathway

Berger, Angela K.; Danthi, Pranav

Activation of NF-B is not required for reovirus-induced cell death. Previously, we have suggested that activation of NF-B early, 6 to 8 h after reovirus infection, is required for caspase-8mediated generation of tBid and attendant activation of effector caspases (14) . To determine if NF-B activation following infection of L929 cells with T1L and T3D differs, we measured the activation of NF-B by measuring the loss of IB␣ inhibitor from the cytoplasm and the consequent translocation of the NF-B RelA/p65 subunit to the nucleus (21, 36) . Immunoblot analysis of IB␣ levels 7.5 h after infection indicates a greater decrease in IB␣ levels following infection with T3D than following infection with T1L ( Fig. 2A) . Consistent with these data, we found a significantly greater level of RelA in the nuclear extracts of T3D-infected cells than in those of T1L-infected cells (Fig. 2B) . To test the idea that the enhanced death-inducing potential of T3D in L929 cells may be related to an increased capacity to activate NF-B, we determined if blockade of NF-B activation would diminish cell death following T3D infection. For these experiments, we assessed the capacity of T3D to promote cell death in cells treated with an IB␣ kinase (IKK) inhibitor, BAY- 65-1942 (21, 37) . We found that blockade of IKK activation had no effect on the induction of cell death by T3D (Fig. 2C ). Treatment of cells with TNF-␣ in the presence of the IKK inhibitor resulted in cell death, demonstrating that the IKK inhibitor is functional in L929 cells and can block TNF-␣-induced prosurvival NF-B signaling. To ensure that the IKK inhibitor also blocked the unusual IKK complex activated by reovirus (21) , we assessed the effect of IKK inhibition on effector caspase activation following reovirus infection. Consistent with the requirement of IKK activity in promoting proapoptotic signaling following reovirus infection (21) , we found that in comparison to dimethyl sulfoxide (DMSO)-treated cells, effector caspase activity following reovirus infection was significantly diminished following treatment of cells with the IKK inhibitor (Fig. 2D) . Thus, despite blocking proapoptotic signaling following reovirus infection, the IKK inhibitor failed to block reovirus-induced cell death. These studies reveal that T3D is capable of activating a cell death pathway that is not dependent on target gene transcription by NF-B. Though this observation matches that from primary cardiac myocytes, it is distinct from that reported for other cell types (20, 34, 38) . The unexpected dispensability of NF-B signaling for cell death in L929 cells prompted us to focus on defining the nature of this cell death pathway.