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Brief Report
15 May 2011

Phosphatidylinositol 3-Kinase Signaling Delays Sendai Virus-Induced Apoptosis by Preventing XIAP Degradation


Sendai virus (SeV) infection causes apoptosis, which is manifested only late after infection; however, inhibition of phosphatidylinositol 3-kinase (PI3K) dramatically accelerates the process. We report here that rapid apoptosis uses the same mitochondrial apoptotic pathway as slow apoptosis. Cytoplasmic cytochrome c (cyt c) was released early in both cases, but the antiapoptotic protein XIAP prevented early activation of the caspases in cells with active PI3K. When the enzyme was inhibited, XIAP was degraded rapidly in infected cells, allowing cyt c to cause caspase activation and early apoptosis. Thus, SeV infection-mediated apoptosis is temporally regulated by the prevention of XIAP degradation by PI3K.


Apoptosis is an integral component of the cellular defense against viruses. Sendai virus (SeV), a member of the paramyxovirus family, induces apoptosis in cultured cells 36 to 48 h after infection (4). Studies have shown that this process requires signaling through retinoic acid-inducible gene I (RIG-I), leading to interaction between interferon regulatory factor 3 (IRF-3) and the proapoptotic protein Bax and translocation of this complex to the mitochondrial membrane. This complex in turn permeabilizes mitochondria, allowing the release of cytochrome c (cyt c) to the cytoplasm and the subsequent activation of caspases 9 and 3 (2), culminating in apoptosis. Cytosolic cyt c triggers apoptosis by activating the apoptosome complex, which consists of multimeric apoptotic protease-activating factor 1 (APAF-1), procaspase 9, procaspase 3 (9), and inhibitor of apoptosis proteins (IAP), such as XIAP, which block procaspase 9 cleavage and activation of the apoptotic cascade (8). In addition to activating IRF-3, SeV activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to phosphorylation of Akt. Inhibiting PI3K signaling, using the chemical inhibitor LY294002 (LY), markedly changes the time course of cell death, with most SeV-plus-LY-treated cells undergoing apoptosis within 6 h of infection (6). In this study, we investigated how PI3K prevents early apoptosis of SeV-infected cells. Our results indicate that even when PI3K is active, the IRF-3/Bax-mediated apoptotic pathway is activated early after SeV infection by proceeding up to the step of cyt c release from the mitochondria. However, the antiapoptotic protein XIAP prevented cyt c-mediated triggering of the downstream steps of apoptosis. When PI3K activity was blocked, XIAP was degraded and early apoptosis of SeV-infected cells proceeded unabated.

Early apoptosis is mediated by caspase 9 activation.

HT1080 cells were treated for 30 min before being infected and during infection with 50 μM LY294002. The cells were infected with SeV at a multiplicity of infection (MOI) of 10. High levels of caspase 3/7 activity, as measured by a fluorescent-substrate cleavage assay, were observed 6 h after SeV infection, but only in LY-treated cells; such activity was not noticeable even after 24 h of SeV infection in untreated cells (Fig. 1 A). By analyzing Western blots of LY-treated infected cells, we observed early activation of caspases 3 and 9 through cleavage of the respective procaspases; cleavage of poly(ADP-ribose) polymerase (PARP), an indicator of apoptosis (Fig. 1B), was also observed. Untreated cells contained higher levels of SeV C protein than treated cells after 6 h of infection, indicating better virus replication in cells with active PI3K (Fig. 1C). Late apoptosis of untreated SeV-infected cells requires activation of caspase 9, normally associated with mitochondrial apoptosis, rather than caspase 8 (2); to determine whether the same was true for early apoptosis of LY-treated cells, we ablated caspase 8 or 9 levels using small interfering RNAs (siRNAs). Apoptosis, measured as PARP cleavage, was blocked, and caspase 3 cleavage was greatly reduced, by caspase 9 siRNA, but neither apoptosis nor caspase 3 cleavage was affected by caspase 8 siRNA (Fig. 1D), supporting the idea that early apoptosis in LY-treated cells is mediated primarily by caspase 9.
Fig. 1.
Fig. 1. PI3K promotes SeV gene expression by blocking caspase 9-mediated early apoptosis of the infected cell. (A) Caspase 3/7 activities in extracts of HT1080 cells infected with SeV for 6 h, infected and LY-treated for 6 h, infected for 24 h, or mock infected for 24 h. (B) Caspase activation and PARP cleavage. Cells were LY treated and/or SeV infected, as indicated, for 6 h, and cellular levels of the indicated proteins were determined by Western blotting. β-Actin served as a loading control. c-PARP, cleaved PARP. (C) SeV C protein levels in cells infected for 6 h in the absence or presence of LY treatment. (D) Cell lines treated with siRNA against caspase 8 (siC8), caspase 9 (siC9), or a nontargeting control (siNT) were infected with SeV and LY treated for 6 h, and the levels of different proteins were determined to confirm the specific knockdown of caspase 8 or caspase 9 and the consequences for caspase 3 and PARP cleavage.

Early apoptosis is mediated by mitochondrial translocation of IRF-3 and Bax and cytoplasmic release of cyt c.

Previously, we have shown that late apoptosis is mediated by a gene induction-independent function of IRF-3, which involves IRF-3-Bax interaction, their cotranslocation to mitochondria, and the resultant cytoplasmic release of cyt c (2). Results presented in Fig. 2 show that the same mechanism also operates for early apoptosis of LY-treated infected cells. IRF-3 was required (Fig. 2A), but new gene induction, which was blocked by treating cells with actinomycin D (1 μg/ml; treatment protocol was the same as for LY294002), was not (Fig. 2B). Bax was also required for early apoptosis, because Bax siRNA-treated cells showed much less PARP cleavage than cells treated with control siRNA (Fig. 2C). To further investigate the mechanism of early apoptosis, we measured the translocation of IRF-3 and Bax from the cytoplasm to mitochondria by purifying mitochondria from cells infected for 2 h. Bax and IRF-3 accumulated in this fraction in treated infected cells (Fig. 2D), further supporting the idea that early and late apoptosis occurs using the same apoptotic pathway. As expected, early mitochondrial translocation of IRF-3 and Bax caused cyt c release to the cytoplasm in LY-treated cells. Surprisingly the same kinetics was also observed for cells that were infected but not treated with LY (Fig. 2E). These observations indicate that PI3K exerts its antiapoptotic effect downstream of mitochondrial permeabilization but upstream of caspase cascade activation, since early caspase activation occurred only in LY-treated cells (Fig. 1).
Fig. 2.
Fig. 2. Early apoptosis is mediated by IRF-3 and Bax translocation to mitochondria and consequent cyt c release in the cytoplasm. (A) Caspase 3/7 activity in control and IRF-3-ablated cells after 6 h of SeV infection and/or LY treatment. (B) PARP cleavage in cells treated with actinomycin D to block cellular mRNA transcription. Cells were infected with SeV for 6 h where indicated; all cells were treated with LY. (C) Inhibition of PARP cleavage in Bax-ablated cells. Cells were infected with SeV for 6 h as indicated, and all cells were treated with LY. (D) Mitochondrial translocation of IRF-3 and Bax. Mitochondria were prepared from LY-treated cells after 6 h of infection or mock infection. Porin, a mitochondrial protein marker, served as the loading control. (E) Release of cyt c to the cytoplasm. Cells were treated for 6 h as indicated, and cytoplasmic fractions were analyzed for the presence of cyt c; β-actin served as the loading control.

Early and late apoptosis requires a reduction in the level of XIAP, an antiapoptotic protein.

In the mitochondrial apoptotic cascade, cytosolic cyt c interacts with APAF-1 (5), facilitating oligomerization of APAF-1, which allows the recruitment of procaspase 9 in a complex called the apoptosome, leading to its autocatalytic cleavage to active caspase 9, which in turn initiates downstream caspase cleavage and apoptosis (9). XIAP binds to procaspase 9 and interferes with its autocatalytic cleavage and activation (8) in the apoptosome; it also blocks the active sites of caspases 3 and 7 (1, 7). To determine whether XIAP aided early survival of untreated SeV-infected cells, XIAP protein levels in LY-treated and untreated cells were compared after 6 h of virus infection; less XIAP was present in LY-treated infected cells (Fig. 3A). This result suggests that SeV infection triggers early degradation of XIAP but that virus-activated PI3K blocks this effect. As expected, when XIAP expression was knocked down by siRNA, early PARP cleavage was observed even without blocking PI3K (Fig. 3F). Even in the presence of PI3K activity, XIAP was degraded in infected cells after 24 h (Fig. 3E). Virus-induced XIAP degradation requires activation of the RIG-I pathway. When we tested the process in HT1080 cells in which RIG-I signaling was blocked by expression of RIG-IC, a dominant negative inhibitor of RIG-I signaling (10), we did not observe XIAP degradation upon infection, even after LY treatment (Fig. 3B). The reduced level of XIAP in early apoptotic cells was reflected in the severely reduced level of the protein in the apoptosome complex, as revealed by its coimmunoprecipitation with APAF-1 from extracts of treated and untreated cells infected for 6 h (Fig. 3C). To establish that the observed reduction in XIAP caused early apoptosis of LY-treated infected cells, we induced high levels of ectopic expression of XIAP by the transient transfection of a pcDNA3-6myc-XIAP plasmid (or vector control). Much less caspase 3 cleavage was observed in infected LY-treated cells overexpressing XIAP than in vector control cells (Fig. 3D). Similarly, in XIAP-overexpressing cells, PARP was not cleaved even 48 h after infection of untreated cells (Fig. 3G). This observation of protection against early apoptosis of LY-treated infected cells expressing a high level of XIAP suggests that the untreated infected cells are indeed protected by PI3K preventing early XIAP degradation.
Fig. 3.
Fig. 3. XIAP levels dictate timing of apoptosis in response to SeV. (A) XIAP levels in cells treated and/or infected for 6 h. (B) XIAP levels in control (HT1080) or RIG-IC-expressing (RIG IC) cells after LY treatment with or without 6 h of infection. (C) Coimmunoprecipitation of XIAP with APAF-1. APAF-1 was immunoprecipitated from cells after 6 h of infection/treatment, and the levels of XIAP and APAF1 in the precipitates were determined by Western blot analysis. (D) Ectopic expression of XIAP blocks early apoptosis. Caspase 3 levels were determined in infected, LY-treated, and infected control (Vector) cells or XIAP-overexpressing (XIAP) cells. Similar results were seen in late apoptosis in response to SeV. (E) XIAP was degraded by 24 h after SeV infection. (F) Cell lines treated with siRNA against XIAP (siX) showed PARP cleavage 6 h after SeV infection, unlike parental HT1080 (HT) cells. (G) Ectopic XIAP expression stops PARP cleavage in response to SeV 48 h after infection.
We have been investigating the nature of the apoptotic response triggered by SeV. Our earlier studies revealed that SeV and many other viruses that activate the cytoplasmic RIG-I-like helicases trigger similar apoptotic responses mediated by the transcription factor IRF-3. However, the apoptotic pathway of IRF-3 is distinct from its transcriptional pathway, although both require beta interferon promoter stimulator 1 (IPS-1), TNF receptor-associated factor 3 (TRAF-3), and TANK binding kinase 1 (TBK-1) (2). Activation of IRF-3 in the apoptotic pathway leads to its binding to Bax, their cotranslocation to the mitochondria, cytoplasmic release of cyt c, activation of caspase 9, and the consequent downstream apoptotic events. Surprisingly, unlike the rapid gene-inducing nuclear action of IRF-3, completion of the full apoptotic process takes more than 24 h after infection. However, if PI3K activity is blocked, apoptosis of the infected cells happens rapidly and all cells are killed within 6 h of infection. The results reported here led us to better understand the PI3K-mediated temporal regulation of the SeV-elicited apoptotic process (Fig. 4). First, we demonstrated that early apoptosis of SeV-infected cells uses the same pathway that we delineated for late apoptosis. It requires similar mitochondrial translocation of IRF-3 and Bax, cyt c release, and caspase 9 activation. Previously, using chemical inhibitors, we concluded that caspase 8 activation is critical for rapid apoptosis, but it appears that this conclusion was wrong. Because the inhibitors previously used are promiscuous and there is cross talk between the caspase 8 and caspase 9 pathways, their distinction is difficult. In this study, siRNA-mediated ablation and further characterization of the pathway clearly establish that the mitochondrial caspase 9 pathway mediates early apoptosis in virus-infected cells in which PI3K is not active. It was surprising to determine that even in the presence of PI3K activity, many early steps of the mitochondrial apoptotic pathway are activated early after virus infection. There was no difference between LY-treated and untreated cells 6 h after infection up to the point of cyt c release from the mitochondria. The critical temporal regulation came after that, at the stage of caspase 9 activation, and this regulation was mediated by the stabilization of XIAP by PI3K signaling. Our study revealed several unexpected features of SeV-induced apoptosis. Virus infection activated at least three pathways, all of which contributed to the apoptotic outcome. First, the IRF-3/Bax apoptotic response was triggered by the RIG-I pathway; second, this pathway also triggered XIAP destabilization. Although there are reports in the literature that XIAP can be degraded faster upon its phosphorylation (3), we do not know at this time whether this was true in our study. The third pathway activated by SeV infection is the PI3K/Akt pathway, which puts brakes on XIAP degradation. Although the biochemical mechanism of the third effect remains to be studied, it is clear that it is required for efficient virus replication (Fig. 1C). It is clear from our results that the timing of apoptosis is critical for efficient virus replication and that the overall process is exquisitely regulated in a temporal fashion by the coordinated activation of several pathways activated by virus infection. Our findings suggest the possibility of therapeutic use of PI3K/Akt inhibitors to control the replication and spread of clinically important viruses.
Fig. 4.
Fig. 4. A model of temporal regulation of apoptosis in SeV-infected cells by PI3K. Virus infection uses the RIG-I pathway to activate cyt c release by IRF-3/Bax translocation to mitochondria. XIAP degradation is also induced by the RIG-I pathway but blocked by a virus-activated PI3K/Akt pathway in the early phase of virus infection. Prevention of XIAP degradation blocked caspase 9 activation in the APAF-1/cyt c apoptosome complex.


The antibody against SeV C protein was a kind gift from Atsushi Kato, University of Tokyo.
This work was supported by grant AI073303 from the National Institutes of Health.


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Information & Contributors


Published In

cover image Journal of Virology
Journal of Virology
Volume 85Number 1015 May 2011
Pages: 5224 - 5227
PubMed: 21367892


Received: 10 January 2011
Accepted: 24 February 2011
Published online: 15 May 2011


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Christine L. White
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio
Saurabh Chattopadhyay
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio
Ganes C. Sen [email protected]
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio

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