Apoptosis-signaling pathways involving death receptors DR4 and DR5 and death ligand TRAIL, as well as Fas and FasL, have been implicated in apoptosis induction by reovirus [17, 42, 43] . However, it is not known which of these pathways mediates cleavage-induced activation of Bid. It is also not understood whether NF-kB regulates the activation of these pathways. Since upregulation of Fas following reovirus infection is dependent on prodeath signaling via c-Jun N terminal kinase (JNK) [43] , and because JNK is activated via a mechanism distinct from NF-kB following reovirus infection [44] , we focused our efforts on assessing the regulation and function of death-receptor signaling via TRAIL following reovirus infection. For these studies, we assessed the capacity of reovirus to induce apoptosis in MEFs lacking TRAIL-R, the only known receptor for TRAIL on murine cells [45, 46, 47] (Figure 4 ). In comparison to mock infection, T3D infection of wild-type cells resulted in an MOI-dependent ,5-to 20-fold increase in caspase-3/7 activity at 24 h post-infection ( Figure 4A ). Although T3D infection of TRAIL-R-deficient cells also resulted in an increase in caspase-3/7 activity in comparison to mock-infection, the magnitude of this increase was only ,2-to 6-fold. Assessment of apoptosis in wild-type and TRAIL-Rdeficient MEFs using AO staining also showed an increase in apoptosis both in wild-type and TRAIL-R-deficient cells in comparison to mock-infected cells ( Figure 4B ). However, a substantially greater fraction of wild-type cells showed morphologic features of apoptosis in comparison to TRAIL-R-deficient cells infected at equivalent MOI, suggesting that efficient induction of apoptosis by reovirus requires TRAIL-R. T3D displayed comparable replication kinetics and produced equivalent yields in wild-type and TRAIL-R-deficient cells ( Figure 4C ). Thus, Figure 2 . Bid is required for apoptosis induction following reovirus infection. (A) Wild-type or Bid-deficient MEFs were adsorbed with T3D at the MOIs shown. After incubation at 37uC for 24 h, caspase-3/7 activity in cell lysates was determined. Results are expressed as the mean ratio of caspase-3/7 activity from infected cell lysates to that from mock-infected cells for triplicate samples. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. Cells were treated with 10 mM staurosporine (Sts) for 24 h as a control. (B) Wild-type or Bid-deficient MEFs were adsorbed with T3D at the MOIs shown. After incubation at 37uC for 48 h, cells were stained with AO. Results are expressed as the mean percentage of cells undergoing apoptosis for three independent experiments. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. (C) Wild-type and Bid-deficient MEFs were adsorbed with 10 4 particles/ cell of T3D. After incubation at 37uC for 18 h, cells were visualized by immunostaining with polyclonal reovirus-specific antiserum, followed by incubation with Alexa546-labeled anti-rabbit IgG. Reovirus-infected cells were quantified by counting fluorescent cells. Results are expressed as mean fluorescent focus units (FFU) per field for triplicate samples. Error bars indicate SD. (D) Wild-type and Bid-deficient MEFs were adsorbed with T3D at an MOI of 2 PFU/cell. The inoculum was removed, and cells were incubated at 37uC for the times shown. Viral titers were determined after two cycles of freeze-thaw by plaque assay using L929 cells. Results are presented as mean titers from three independent experiments. Error bars indicate SD. doi:10.1371/journal.ppat.1000980.g002 differences in the apoptotic potential of reovirus in wild-type and TRAIL-R-deficient cells are not associated with differences in reovirus growth in these cells.