Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels - PubMed (original) (raw)

Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels

Douglas J Kominsky et al. J Virol. 2002 Nov.

Abstract

Many viruses belonging to diverse viral families with differing structure and replication strategies induce apoptosis both in cultured cells in vitro and in tissues in vivo. Despite this fact, little is known about the specific cellular apoptotic pathways induced during viral infection. We have previously shown that reovirus-induced apoptosis of HEK cells is initiated by death receptor activation but requires augmentation by mitochondrial apoptotic pathways for its maximal expression. We now show that reovirus infection of HEK cells is associated with selective cytosolic release of the mitochondrial proapoptotic factors cytochrome c and Smac/DIABLO, but not the release of apoptosis-inducing factor. Release of these factors is not associated with loss of mitochondrial transmembrane potential and is blocked by overexpression of Bcl-2. Stable expression of caspase-9b, a dominant-negative form of caspase-9, blocks reovirus-induced caspase-9 activation but fails to significantly reduce activation of the key effector caspase, caspase-3. Smac/DIABLO enhances apoptosis through its action on cellular inhibitor of apoptosis proteins (IAPs). Reovirus infection is associated with selective down-regulation of cellular IAPs, including c-IAP1, XIAP, and survivin, effects that are blocked by Bcl-2 expression, establishing the dependence of IAP down-regulation on mitochondrial events. Taken together, these results are consistent with a model in which Smac/DIABLO-mediated inhibition of IAPs, rather than cytochrome c-mediated activation of caspase-9, is the key event responsible for mitochondrial augmentation of reovirus-induced apoptosis. These studies provide the first evidence for the association of Smac/DIABLO with virus-induced apoptosis.

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Figures

FIG. 1.

FIG. 1.

Smac/DIABLO and cytochrome c are present in the cytosol of reovirus-infected cells. HEK 293 lysates were prepared at the indicated time points from mock-infected cells, reovirus-infected cells, or Bcl-2-overexpressing reovirus-infected cells and resolved using SDS-PAGE. Western blot analysis was performed using anti-Smac antibodies (A) and anti-cytochrome c antibodies (B). The Western is representative of two separate experiments. Lanes c, cytoplasmic fraction; lanes m, mitochondrial fraction. h.p.i., hours postinfection.

FIG. 2.

FIG. 2.

Reovirus infection does not induce the mitochondrial release of AIF. HEK 293 lysates were prepared at the indicated time points from mock-infected and reovirus-infected cells and resolved using SDS-PAGE. Blots were probed with anti-AIF antibodies and are representative of three separate experiments. Lanes c, cytoplasmic fraction; lanes m, mitochondrial fraction. h.p.i., hours postinfection.

FIG. 3.

FIG. 3.

Mitochondrial ΔΨm is not altered in reovirus-infected cells. HEK 293 cells were mock-infected (A) or infected with reovirus for 0 h (B), 6 h (C), 12 h (D), or 24 h (E). Control cells were treated with valinomycin (panel F). Cells were incubated with the membrane potential-sensitive dye JC-1 and analyzed using flow cytometry. Red fluorescence indicates mitochondria with intact ΔΨm and green fluorescence indicates loss of ΔΨm. Results are representative of three separate experiments.

FIG. 4.

FIG. 4.

Inhibition of caspase-9 activation does not prevent effector caspase activation. Western blot analysis was performed using HEK 293 lysates harvested at the indicated time points from mock-infected and reovirus-infected cells and probed with anti-caspase-9 antibodies (A) or anti-PARP antibodies (C). Control lanes represent Jurkat cell lysates untreated (−) or treated (+) with activating anti-Fas antibody and harvested at 8 h posttreatment. Each Western blot is representative of two separate experiments. (B) Fluorogenic substrate assays were performed in triplicate. Error bars represent standard error of the mean. Fluorescence is expressed as arbitrary units. h.p.i., hours postinfection.

FIG. 4.

FIG. 4.

Inhibition of caspase-9 activation does not prevent effector caspase activation. Western blot analysis was performed using HEK 293 lysates harvested at the indicated time points from mock-infected and reovirus-infected cells and probed with anti-caspase-9 antibodies (A) or anti-PARP antibodies (C). Control lanes represent Jurkat cell lysates untreated (−) or treated (+) with activating anti-Fas antibody and harvested at 8 h posttreatment. Each Western blot is representative of two separate experiments. (B) Fluorogenic substrate assays were performed in triplicate. Error bars represent standard error of the mean. Fluorescence is expressed as arbitrary units. h.p.i., hours postinfection.

FIG. 4.

FIG. 4.

Inhibition of caspase-9 activation does not prevent effector caspase activation. Western blot analysis was performed using HEK 293 lysates harvested at the indicated time points from mock-infected and reovirus-infected cells and probed with anti-caspase-9 antibodies (A) or anti-PARP antibodies (C). Control lanes represent Jurkat cell lysates untreated (−) or treated (+) with activating anti-Fas antibody and harvested at 8 h posttreatment. Each Western blot is representative of two separate experiments. (B) Fluorogenic substrate assays were performed in triplicate. Error bars represent standard error of the mean. Fluorescence is expressed as arbitrary units. h.p.i., hours postinfection.

FIG. 5.

FIG. 5.

Reovirus infection leads to the cleavage of XIAP. Western blot analysis was performed using HEK 293 lysates harvested at the indicated time points from mock-infected, reovirus-infected, and Bcl-2-overexpressing reovirus-infected cells and probed with anti-XIAP antibodies. Control lanes represent Jurkat cell lysates untreated (−) or treated (+) with activating anti-Fas antibody and harvested at 8 h posttreatment. The Western blot is representative of three separate experiments. h.p.i., hours postinfection.

FIG. 6.

FIG. 6.

Cellular levels of survivin are reduced following reovirus infection. Western blot analysis was performed using HEK 293 lysates harvested at the indicated time points from mock-infected, reovirus-infected, and Bcl-2-overexpressing reovirus-infected cells and probed with antisurvivin antibodies and antiactin to demonstrate equal protein loading. Control lanes represent Jurkat cell lysates untreated (−) or treated (+) with activating anti-Fas antibody and harvested at 8 h posttreatment. The Western blot is representative of two separate experiments. h.p.i., hours postinfection.

FIG. 7.

FIG. 7.

Reovirus infection induces a reduction in c-IAP1 protein level. Western blot analysis was performed using HEK 293 lysates harvested at the indicated time points from mock-infected, reovirus-infected, and Bcl-2-overexpressing reovirus-infected cells and probed with anti-c-IAP1 antibodies and antiactin to demonstrate equal protein loading. Control lanes represent Jurkat cell lysates untreated (−) or treated (+) with activating anti-Fas antibody and harvested at 8 h posttreatment. The Western blot is representative of two separate experiments. h.p.i., hours postinfection.

FIG. 8.

FIG. 8.

Cellular c-IAP2 protein levels are unaffected following reovirus infection. Western blot analysis was performed using HEK 293 lysates harvested at the indicated time points from mock-infected, reovirus-infected, and Bcl-2-overexpressing reovirus-infected cells and probed with anti-c-IAP2 antibodies and antiactin to demonstrate equal protein loading. Control lanes represent Jurkat cell lysates untreated (−) or treated (+) with activating anti-Fas antibody and harvested at 8 h posttreatment. The Western blot is representative of two separate experiments. h.p.i., hours postinfection.

FIG. 9.

FIG. 9.

Model of cellular pathways involved in reovirus-induced apoptosis in HEK cells. Reovirus infection leads to the activation of caspase-8 and cleavage of the proapoptotic molecule Bid. These events promote the release of Smac/DIABLO and cytochrome c from the mitochondria of infected cells. The release of these proteins can be prevented by the overexpression of Bcl-2. Cytosolic Smac/DIABLO inhibits the antiapoptotic effect exerted by XIAP, survivin, and c-IAP1, leading to the sustained activation of both initiator caspases and the effector caspase, caspase-3. While caspase-9 is activated and undoubtedly participates in reovirus-induced apoptosis, its activation appears to be dispensable for this process.

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