Reovirus-induced Apoptosis Research Articles

Down-regulation of HIF-1α by oncolytic reovirus infection independently of VHL and p53

Many oncolytic viruses are currently being tested as potential cancer therapeutic agents. To be effective, these viruses must replicate and propagate efficiently through the tumor mass. However, it is possible that the hypoxia that characterizes many tumors may be an obstacle to viral therapy because of its inhibition of viral replication and propagation. We, therefore, decided to test how oncolytic reovirus and its target cells respond to hypoxia. We found that reovirus infection suppresses hypoxia inducible factor (HIF)-1alpha protein levels (but not transcript abundance) in colon cancer HCT116 cells under CoCl(2) or hypoxia. Reovirus infection was able to reduce HIF-1alpha levels in both von Hippel Lindau (VHL)-/- renal carcinoma A498 and p53-/- HCT116 cells, indicating that the decrease of HIF-1alpha mediated by reovirus requires neither VHL nor p53 proteins. However, treatment with the inhibitor MG132 restored HIF-1alpha levels, suggesting that reovirus-induced HIF-1alpha decrease needs proteosomal activity. A498 VHL-/- cells with constitutive expression of HIF-1alpha were relatively resistant to reovirus-induced apoptosis when compared with A498 VHL+/+ cells. However, we found that the use of YC-1 to target HIF-1alpha promoted reovirus-induced apoptosis in A498 VHL-/- cells. Accordingly, we propose that reovirus may be used together with YC-1 as a potential therapeutic agent against chemoresistant or radioresistant tumors that are hypoxic and show increased levels of HIF-1alpha.

Inhibition of GSK-3β enhances reovirus-induced apoptosis in colon cancer cells

Reovirus functions as an oncolytic agent for many types of cancer including colon cancer. Although most studies have emphasized the role of activated Ras signaling in enhancing reoviral oncolysis in susceptible cells, we note that many colon cancers also display elevated beta-catenin. Thus, it is possible that enhanced beta-catenin may augment reoviral susceptibility in colon cancer cells. To explore this hypothesis, HEK293 cells were treated with the glycogen synthase kinase (GSK)-3beta inhibitor LiCl, thereby inducing beta-catenin, followed by reoviral infection. Co-administration with LiCl indeed enhanced cell death compared to reovirus infection alone, but this was not associated with elevated reoviral replication. Similarly, HEK293 cells expressing the Frizzled-1 receptor in Wnt3a-conditioned medium also showed reovirus replication equivalent to that in cells in control medium, further suggesting that up-regulation of beta-catenin does not enhance the replication of reovirus. Instead, we observed that inhibition of GSK-3beta with LiCl decreased reovirus-induced NF-kappaB activation, leading to accelerated apoptosis via caspase 8 activation. We further found that colon cancer HCT116 cells were sensitized to apoptosis by co-treatment with reovirus and a GSK-3beta inhibitor, AR-A014418. Finally, we identified that inhibition of NF-kappaB sensitized apoptosis of HEK293 or HCT 116 cells during reovirus infection. Taken together, we propose that inhibition of GSK-3beta sensitizes reovirus-induced apoptosis of colon cancer cells by down-regulation of NF-kappaB activity, offering a potentially improved therapeutic strategy for the treatment of colon cancer.

Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the μ1 φ Domain

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein μ1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of μ1 indicates that the μ1 ϕ domain is sufficient to elicit a cell death response. To evaluate the contribution of the μ1 ϕ domain to the induction of apoptosis following reovirus infection, ϕ mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in ϕ diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in ϕ affect the apoptotic potential of reovirus, suggesting that ϕ initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective ϕ mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the ϕ domain of μ1 plays an important regulatory role in reovirus-induced apoptosis and disease.

Hepatitis C Virus Triggers Apoptosis of a Newly Developed Hepatoma Cell Line Through Antiviral Defense System

Hepatitis C virus (HCV) has a tendency to cause chronic viral infection. Viral evasion of host immune systems plays a key role in the pathogenesis of HCV. However, the interaction between HCV and hepatocyte innate antiviral defense systems is not understood. The aim of this study was to examine how human hepatocytes respond to HCV infection. We have established a novel human hepatoma cell line, LH86, from a well-differentiated hepatocellular carcinoma tissue. An infectious HCV isolate, JFH-1, was used to infect LH86 cells. HCV replication and apoptosis after viral infection were examined. Mechanisms of HCV-induced apoptosis were determined. Type I interferon induction and the relevant signaling molecules were examined. LH86 cells permitted JFH-1 HCV infection. The viral infection caused massive apoptosis. The apoptosis was related to viral replication, because blocking viral entry with anti-CD81 or suppressing viral replication with interferon protected cells from HCV-induced apoptosis. The HCV-induced apoptosis appeared to be triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, death receptor 4 and death receptor 5, which were up-regulated in HCV infection. HCV also activated interferon response factor 3, which induced expression of interferon and TRAIL in LH86 cells. Our study showed that a specific HCV isolate, JFH-1, is cytopathic in this new hepatoma cell line. LH86 cells mount an intact innate antiviral defense through induction of interferon and triggering apoptosis of infected cells. This study reveals a novel mechanism by which host hepatocytes respond to acute HCV infection.

Retinoic Acid-inducible Gene-I and Interferon-β Promoter Stimulator-1 Augment Proapoptotic Responses Following Mammalian Reovirus Infection via Interferon Regulatory Factor-3

During viral infection, cells initiate antiviral responses to contain replication and inhibit virus spread. One protective mechanism involves activation of transcription factors interferon regulatory factor-3 (IRF-3) and NF-kappaB, resulting in secretion of the antiviral cytokine, interferon-beta. Another is induction of apoptosis, killing the host cell before virus disseminates. Mammalian reovirus induces both interferon-beta and apoptosis, raising the possibility that both pathways are initiated by a common cellular sensor. We show here that reovirus activates IRF-3 with kinetics that parallel the activation of NF-kappaB, a known mediator of reovirus-induced apoptosis. Activation of IRF-3 requires functional retinoic acid inducible gene-I and interferon-beta promoter stimulator-1, but these intracellular sensors are dispensable for activation of NF-kappaB. Interferon-beta promoter stimulator-1 and IRF-3 are required for efficient apoptosis following reovirus infection, suggesting a common mechanism of antiviral cytokine induction and activation of the cell death response.

Avian reovirus-induced apoptosis related to tissue injury

Apoptosis plays an important role in pathogenesis of many viral infections. Infection of chicken with avian reovirus S1133 causes tissue injury related to virus-induced apoptosis. To determine whether avian reovirus (ARV) induced apoptosis in chicken tissues, six 3-week-old specific pathogen free White Leghorn chicks were inoculated with ARV S1133. Tissues were dual-labelled for the simultaneous detection of viral antigen containing and apoptotic cells. DNA laddering was detected in ARV-infected but not mock-infected chicken tissues. Dual-labelling assay revealed that the majority of antigen-expressing cells were not apoptotic. Surprisingly, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. Syncytium formation in ARV-infected chicken tissues undergoing apoptosis was apparent, suggesting a correlation between virus replication and apoptosis in chicken tissues.

IκB Kinase Subunits α and γ Are Required for Activation of NF-κB and Induction of Apoptosis by Mammalian Reovirus

Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-kappaB and downstream cellular genes. To better understand the mechanism of NF-kappaB activation by reovirus, NF-kappaB signaling intermediates under reovirus control were investigated at the level of Rel, IkappaB, and IkappaB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This biphasic pattern of Rel protein activation is associated with the degradation of the NF-kappaB inhibitor IkappaBalpha but not the structurally related inhibitors IkappaBbeta or IkappaBepsilon. Using IKK subunit-specific small interfering RNAs and cells deficient in individual IKK subunits, we demonstrate that IKKalpha but not IKKbeta is required for reovirus-induced NF-kappaB activation and apoptosis. Despite the preferential usage of IKKalpha, both NF-kappaB activation and apoptosis were attenuated in cells lacking IKKgamma/Nemo, an essential regulatory subunit of IKKbeta. Moreover, deletion of the gene encoding NF-kappaB-inducing kinase, which is known to modulate IKKalpha function, had no inhibitory effect on either response in reovirus-infected cells. Collectively, these findings indicate a novel pathway of NF-kappaB/Rel activation involving IKKalpha and IKKgamma/Nemo, which together mediate the expression of downstream proapoptotic genes in reovirus-infected cells.

Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.

JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis.

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein sigma1 and membrane penetration protein mu1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a sigma1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L x T3D reassortant viruses revealed that the mu1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of sigma1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the mu1 protein plays an essential role in stimulating proapoptotic signaling.

Sensitization to Apoptosis Underlies Kras D12 -Dependent Oncolysis of Murine C26 Colorectal Carcinoma Cells by Reovirus T3D

Reovirus T3D is an oncolytic agent that preferentially targets tumor cells expressing an activated Ras oncogene. Ras signaling interferes with the cellular stress response that inhibits translation of reovirus RNAs. Murine C26 colorectal carcinoma cells express a mutant KrasD12 gene. Reovirus T3D efficiently kills C26 cells, but not C26 cells in which the KrasD12 mRNA is stably repressed by expression of KrasD12-directed short-hairpin RNAs. Surprisingly, neither reovirus T3D protein synthesis nor T3D virus yields were suppressed by deletion of KrasD12. Rather, reovirus-induced tumor cell apoptosis was completely abrogated as a result of Kras knockdown. We conclude that sensitization of C26 tumor cells to reovirus-induced apoptosis underlies the Ras dependency of reovirus T3D oncolysis.

Organ-specific roles for transcription factor NF-κB in reovirus-induced apoptosis and disease

Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-kappaB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-kappaB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-beta mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-beta substantially diminished reovirus replication and apoptosis, which suggests that IFN-beta induction by NF-kappaB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-kappaB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Inhibition of NF-kappa B activity and cFLIP expression contribute to viral-induced apoptosis.

Virus-induced activation of nuclear factor-kappa B (NF-kappaB) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-kappaB is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-kappaB activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-kappaB regulation, where NF-kappaB activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-kappaB activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-kappaB is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-kappaB also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-kappaB-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-kappaB activation in reovirus-infected cells. Further, inhibition of NF-kappaB activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-kappaB and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis.

Mechanisms of Reovirus-Induced Cell Death and Tissue Injury: Role of Apoptosis and Virus-Induced Perturbation of Host-Cell Signaling and Transcription Factor Activation

Reoviruses have provided insight into the roles played by specific viral genes and the proteins they encode in virus-induced cell death and tissue injury. Apoptosis is a major mechanism of cell death induced by reoviruses. Reovirus-induced apoptosis involves both death-receptor and mitochondrial cell death pathways. Reovirus infection is associated with selective activation of mitogen activated protein kinase (MAPK) cascades including JNK/SAPK. Infection also perturbs transcription factor signaling resulting in the activation of c-Jun and initial activation followed by strain-specific inhibition of NF-kappaB. Infection results in changes in the expression of genes encoding proteins involved in cell cycle regulation, apoptosis, and DNA damage and repair processes. Apoptosis is a major mechanism of reovirus-induced injury to key target organs including the CNS and heart. Inhibition of apoptosis through the use of caspase or calpain inhibitors, minocycline, or in caspase 3(-/-) mice all reduce virus-associated tissue injury and enhance survival of infected animals. Reoviruses induce apoptotic cell death (oncolysis) in a wide variety of cancer cells and tumors. The capacity of reoviruses to grow efficiently in transformed cells is enhanced by the presence of an activated Ras signaling pathway likely through mechanisms involving inhibition of antiviral PKR signaling and activation of Ras/RalGEF/p38 pathways. The potential of reovirus-induced oncolysis in therapy of human cancers is currently being investigated in phase I/II clinical trials.

Nonstructural protein sigma1s is a determinant of reovirus virulence and influences the kinetics and severity of apoptosis induction in the heart and central nervous system.

The mechanisms by which viruses kill susceptible cells in target organs and ultimately produce disease in the infected host remain poorly understood. Dependent upon the site of inoculation and strain of virus, experimental infection of neonatal mice with reoviruses can induce fatal encephalitis or myocarditis. Reovirus-induced apoptosis is a major mechanism of tissue injury, leading to disease development in both the brain and heart. In cultured cells, differences in the capacity of reovirus strains to induce apoptosis are determined by the S1 gene segment, which also plays a major role as a determinant of viral pathogenesis in both the heart and the central nervous system (CNS) in vivo. The S1 gene is bicistronic, encoding both the viral attachment protein sigma-1 and the nonstructural protein sigma-1-small (sigma1s). Although sigma1s is dispensable for viral replication in vitro, we wished to investigate the expression of sigma1s in the infected heart and brain and its potential role in reovirus pathogenesis in vivo. Two-day-old mice were inoculated intramuscularly or intracerebrally with either sigma1s(-) or sigma1s(+) reovirus strains. While viral replication in target organs did not differ between sigma1s(-) and sigma1s(+) viral strains, virus-induced caspase-3 activation and resultant histological tissue injury in both the heart and brain were significantly reduced in sigma1s(-) reovirus-infected animals. These results demonstrate that sigma1s is a determinant of the magnitude and extent of reovirus-induced apoptosis in both the heart and CNS and thereby contributes to reovirus pathogenesis and virulence.

JNK Regulates the Release of Proapoptotic Mitochondrial Factors in Reovirus-Infected Cells

Reovirus-induced apoptosis is associated with activation of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) and the JNK-associated transcription factor c-Jun. Here we show that reovirus-induced apoptosis and activation of caspase 3 are inhibited in cells deficient in MEK kinase 1, an upstream activator of JNK in reovirus-infected cells. Inhibition of JNK activity following reovirus infection delays the release of proapoptotic mitochondrial factors and the subsequent onset of apoptosis. In contrast, reovirus-induced apoptosis is not blocked by infection with adenovirus expressing dominant-negative c-Jun, and c-Jun activation does not correlate with apoptosis in reovirus-infected cells. This is the first report demonstrating that JNK is associated with regulation of mitochondrial pathways of apoptosis following viral infection.

Reovirus-induced alteration in expression of apoptosis and DNA repair genes with potential roles in viral pathogenesis.

Reoviruses are a leading model for understanding cellular mechanisms of virus-induced apoptosis. Reoviruses induce apoptosis in multiple cell lines in vitro, and apoptosis plays a key role in virus-induced tissue injury of the heart and brain in vivo. The activation of transcription factors NF-kappaB and c-Jun are key events in reovirus-induced apoptosis, indicating that new gene expression is critical to this process. We used high-density oligonucleotide microarrays to analyze cellular transcriptional alterations in HEK293 cells after infection with reovirus strain T3A (i.e., apoptosis inducing) compared to infection with reovirus strain T1L (i.e., minimally apoptosis inducing) and uninfected cells. These strains also differ dramatically in their potential to induce apoptotic injury in hearts of infected mice in vivo-T3A is myocarditic, whereas T1L is not. Using high-throughput microarray analysis of over 12,000 genes, we identified differential expression of a defined subset of genes involved in apoptosis and DNA repair after reovirus infection. This provides the first comparative analysis of altered gene expression after infection with viruses of differing apoptotic phenotypes and provides insight into pathogenic mechanisms of virus-induced disease.

Two Distinct Phases of Virus-induced Nuclear Factor κB Regulation Enhance Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis in Virus-infected Cells

Cellular transcription factors are often utilized by infecting viruses to promote viral growth and influence cell fate. We have previously shown that nuclear factor kappaB (NF-kappaB) is activated after reovirus infection and that this activation is required for virus-induced apoptosis. In this report we identify a second phase of reovirus-induced NF-kappaB regulation. We show that at later times post-infection NF-kappaB activation is blocked in reovirus-infected cells. This results in the termination of virus-induced NF-kappaB activity and the inhibition of tumor necrosis factor alpha and etoposide-induced NF-kappaB activation in infected cells. Reovirus-induced inhibition of NF-kappaB activation occurs by a mechanism that prevents IkappaBalpha degradation and that is blocked in the presence of the viral RNA synthesis inhibitor, ribavirin. Reovirus-induced apoptosis is mediated by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a variety of epithelial cell lines. Herein we show that ribavirin inhibits reovirus-induced apoptosis in TRAIL-resistant HEK293 cells and prevents the ability of reovirus infection to sensitize TRAIL-resistant cells to TRAIL-induced apoptosis. Furthermore, TRAIL-induced apoptosis is enhanced in HEK293 cells expressing IkappaBDeltaN2, which blocks NF-kappaB activation. These results indicate that the ability of reovirus to inhibit NF-kappaB activation sensitizes HEK293 cells to TRAIL and facilitates virus-induced apoptosis in TRAIL-resistant cells. Our findings demonstrate that two distinct phases of virus-induced NF-kappaB regulation are required to efficiently activate host cell apoptotic responses to reovirus infection.

Reovirus-induced apoptosis: A minireview.

Reoviruses infect a variety of mammalian hosts and serve as an important experimental system for studying the mechanisms of virus-induced injury. Reovirus infection induces apoptosis in cultured cells in vitro and in target tissues in vivo, including the heart and central nervous system (CNS). In epithelial cells, reovirus-induced apoptosis involves the release of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) from infected cells and the activation of TRAIL-associated death receptors (DRs) DR4 and DR5. DR activation is followed by activation of caspase 8, cleavage of Bid, and the subsequent release of pro-apoptotic mitochondrial factors. By contrast, in neurons, reovirus-induced apoptosis involves a wider array of DRs, including TNFR and Fas, and the mitochondria appear to play a less critical role. These results show that reoviruses induce apoptotic pathways in a cell and tissue specific manner. In vivo there is an excellent correlation between the location of viral infection, the presence of tissue injury and apoptosis, indicating that apoptosis is a critical mechanism by which disease is triggered in the host. These studies suggest that inhibition of apoptosis may provide a novel strategy for limiting virus-induced tissue damage following infection.

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

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.

Reovirus-induced apoptosis requires both death receptor- and mitochondrial-mediated caspase-dependent pathways of cell death.

Apoptosis plays an important role in the pathogenesis of many viral infections. Despite this fact, the apoptotic pathways triggered during viral infections are incompletely understood. We now provide the first detailed characterization of the pattern of caspase activation following infection with a cytoplasmically replicating RNA virus. Reovirus infection of HEK293 cells results in the activation of caspase-8 followed by cleavage of the pro-apoptotic protein Bid. This initiates the activation of the mitochondrial apoptotic pathway leading to release of cytochrome c and activation of caspase-9. Combined activation of death receptor and mitochondrial pathways results in downstream activation of effector caspases including caspase-3 and caspase-7 and cleavage of cellular substrates including PARP. Apoptosis is initiated by death receptor pathways but requires mitochondrial amplification producing a biphasic pattern of caspase-8, Bid, and caspase-3 activation.