Reovirus activates a caspase-independent cell death pathway

Abstract

Virus-induced apoptosis is thought to be the primary mechanism of cell death following reovirus infection. Induction of cell death following reovirus infection is initiated by the incoming viral capsid proteins during cell entry and occurs via NF-κB-dependent activation of classical apoptotic pathways. Prototype reovirus strain T3D displays a higher cell-killing potential than strain T1L. To investigate how signaling pathways initiated by T3D and T1L differ, we methodically analyzed cell death pathways activated by these two viruses in L929 cells. We found that T3D activates NF-κB, initiator caspases, and effector caspases to a significantly greater extent than T1L. Surprisingly, blockade of NF-κB or caspases did not affect T3D-induced cell death. Cell death following T3D infection resulted in a reduction in cellular ATP levels and was sensitive to inhibition of the kinase activity of receptor interacting protein 1 (RIP1). Furthermore, membranes of T3D-infected cells were compromised. Based on the dispensability of caspases, a requirement for RIP1 kinase function, and the physiological status of infected cells, we conclude that reovirus can also induce an alternate, necrotic form of cell death described as necroptosis. We also found that induction of necroptosis requires synthesis of viral RNA or proteins, a step distinct from that necessary for the induction of apoptosis. Thus, our studies reveal that two different events in the reovirus replication cycle can injure host cells by distinct mechanisms.

Importance: Virus-induced cell death is a determinant of pathogenesis. Mammalian reovirus is a versatile experimental model for identifying viral and host intermediaries that contribute to cell death and for examining how these factors influence viral disease. In this study, we identified that in addition to apoptosis, a regulated form of cell death, reovirus is capable of inducing an alternate form of controlled cell death known as necroptosis. Death by this pathway perturbs the integrity of host membranes and likely triggers inflammation. We also found that apoptosis and necroptosis following viral infection are activated by distinct mechanisms. Our results suggest that host cells can detect different stages of viral infection and attempt to limit viral replication through different forms of cellular suicide. While these death responses may aid in curbing viral spread, they can also exacerbate tissue injury and disease following infection.

FIG 1

T3D and T1L differ in their efficiency of cell death induction and effector caspase activation. (A) ATCC L929 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3D or T1L. Following incubation at 37°C for 48 h, cells were stained with AOEB. The results are expressed as the mean percentages of cells undergoing cell death for three independent experiments. Error bars indicate standard deviation (SD). *, P value of <0.05 as determined by Student’s t test in comparison to cells infected with T3D. (B) ATCC L929 cells were adsorbed with T3D or T1L at an MOI of 10 PFU/cell or with PBS (mock). After incubation at 37°C for 24 h, caspase-3/7 activity in cell lysates was determined. Results are expressed as the mean ratios of caspase-3/7 activity from infected cell lysates to that from mock-infected cells for triplicate samples. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to cells infected with T3D.

FIG 2

NF-κB is activated but dispensable for induction of cell death. (A) ATCC L929 cells were adsorbed with PBS (mock) or 100 PFU/cell of T3D or T1L. Following incubation at 37°C for 7.5 h, cytoplasmic extracts were immunoblotted using an antiserum specific for IκBα or tubulin. (B) ATCC L929 cells were adsorbed with PBS (mock) or 100 PFU/cell of T3D or T1L. Following incubation at 37°C for 7.5 h, nuclear extracts were immunoblotted using an antiserum specific for RelA or PSTAIR. (C) ATCC L929 cells were adsorbed with PBS (mock) or T3D at an MOI of 10 PFU/cell or treated with 10 ng/ml TNF-α. After incubation at 37°C for 48 h (24 h for TNF-α), in the presence of DMSO or a 5 µM concentration of the IKK inhibitor BAY-65-1942, the cells were stained with AOEB. The results are expressed as the mean percentages of cells undergoing cell death for three independent experiments. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to cells treated with TNF-α. (D) ATCC L929 cells were adsorbed with PBS (mock) or T3D at an MOI of 10 PFU/cell in the presence of DMSO or a 5 µM concentration of the IKK inhibitor BAY-65-1942. After incubation at 37°C for 24 h, caspase-3/7 activity in cell lysates was determined. Results are expressed as the mean ratios of caspase-3/7 activity from infected cell lysates to that from equivalently treated mock-infected cells for triplicate samples. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to T3D-infected cells treated with DMSO.

FIG 3

Caspase activity is not required for T3D-induced cell death. (A) ATCC L929 cells were adsorbed with PBS (mock) or T3D at an MOI of 10 PFU/cell. After incubation at 37°C for 48 h in the presence of DMSO or 25 µM caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK), or pan-caspase [Z-VAD(OMe)-FMK] inhibitor, cells were stained with AOEB. The results are expressed as the mean percentages of cells undergoing cell death for three independent experiments. Error bars indicate SD. (B) ATCC L929 cells were adsorbed with PBS (mock) or T3D at the MOI of 10 PFU/cell. After incubation at 37°C for 24 h in the presence of DMSO or 25 µM concentrations of each caspase inhibitor, caspase-3/7 activity in cell lysates was determined. Results are expressed as the mean ratios of caspase-3/7 activity from infected cell lysates to that from equivalently treated mock-infected cells for triplicate samples. We note that the DMSO-treated cells are the same as those used in Fig. 2. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to T3D-infected cells treated with DMSO.

FIG 4

T3D-induced cell death exhibits characteristics of necroptosis. (A) ATCC L929 cells were adsorbed with T3D at an MOI of 10 PFU/cell. After incubation at 37°C for 48 h in the presence of DMSO, 50 µM RIP1 inhibitor (Nec-1), or a combination of 50 µM RIP1 inhibitor (Nec-1) and 25 µM pan-caspase inhibitor [Z-VAD(OMe)-FMK], cells were harvested and stained with AOEB. The results are expressed as the mean percentages of cells undergoing cell death for three independent experiments. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to T3D-infected cells treated with DMSO. (B) ATCC L929 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3D. Following incubation at 37°C for the indicated amount of time, ATP levels in cells treated with DMSO, pan-caspase inhibitor, or RIP1 inhibitor were measured. Results are expressed as the mean ratios of ATP from mock-infected cells to that from equivalently treated T3D-infected cells for triplicate samples. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to T3D-infected cells treated with DMSO. (C) ATCC L929 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3D. Following incubation at 37°C for the indicated amount of time, DNase-treated medium from infected cells was resolved by SDS-PAGE and immunoblotted using an antiserum specific for HMGB1. An ~200-kDa band of unknown origin found in medium from infected cells, resolved by SDS-PAGE, and stained using Coomassie blue staining was used as a loading control (LC). Cells treated with TNF-α and pan-caspase inhibitor were used as a necroptosis control.

FIG 5

Viral RNA and protein synthesis are required for induction of necroptosis. (A) ATCC L929 cells were adsorbed with PBS (mock) or an equivalent number of genome-containing (T3D) or genome-deficient, top-component (T3D-TC) virus particles. After incubation at 37°C for 24 h, cells were stained with AOEB. The results are expressed as the mean percentages of cells undergoing cell death for three independent experiments. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to T3D-infected cells at an equivalent MOI. (B) ATCC L929 cells were adsorbed with PBS (mock) or T3D at indicated MOIs or with UV-inactivated T3D (T3D-UV) at equivalent MOIs before irradiation. After incubation at 37°C for 24 h, cells were stained with AOEB. The results are expressed as the mean percentages of cells undergoing cell death for three independent experiments. Error bars indicate SD. *, P value of <0.05 as determined by Student’s t test in comparison to T3D-infected cells at an equivalent MOI.