Reovirus Activates a Caspase-Independent Cell Death Pathway

Berger, Angela K.; Danthi, Pranav

Cells undergoing all forms of necrosis, including necroptosis, are characterized by a depletion of cellular ATP levels (44, 45) . To determine whether ATP levels were affected in T3D-infected cells, we compared ATP levels over a time course of infection using a chemiluminescent assay. While an~35% to 40% reduction in ATP was observed at 24 h after infection with T3D,~75% and 95% of the ATP was lost in T3D-infected cells at 36 and 48 h after infection, respectively (Fig. 4B) . Moreover, the rate at which loss of ATP occurred in T3D-infected cells was diminished by treatment of cells with Nec-1 but not by treatment with a pan-caspase inhibitor. These data provide further support for the idea that T3D-infected L929 cells undergo necroptosis following reovirus infection. Necroptosis alters cellular architecture quite differently from apoptosis. In particular, necroptosis damages the integrity of the plasma membrane. To assess whether plasma membrane integrity was compromised following infection with T3D, we evaluated whether high-mobility group box 1 protein (HMGB1) was released into the medium of infected cells. This protein is not released from apoptotic cells undergoing secondary necrosis, and therefore, leakage of this chromatin-associated protein from cells is considered to be a marker for necrosis (46, 47) . We found that HMGB1 was released into the medium at 24 h following infection. A greater level of HMGB1 was detected in the medium at 36 and 48 h after infection with T3D (Fig. 4C) . Along with the capacity of EB to gain access and stain the nuclei of unfixed, reovirus-infected cells, a measurement equivalent to propidium iodide or sytox staining of necrotic cells (48) , the data presented here indicate that the L929 cell plasma membrane is leaky following infection with T3D. Based on the dispensability of caspases, the requirement for RIP1 kinase activity, the dramatic loss in cellular ATP levels, and the release of HMGB1 from infected cells, we conclude that reo-virus is capable of inducing an alternate cell death pathway, which has been described as necroptosis.