Viruses can trigger the defensive apoptotic response of the infected host cell by a variety of mechanisms, but many viruses possess different tools to overcome this defense (48
). The results of this study allowed us to identify the major apoptotic pathway activated upon poliovirus infection and to ascertain which part of this pathway is targeted by the viral antiapoptotic activity.
The efflux of cytochrome c
from mitochondria and the degradation of procaspase-9 in the poliovirus-infected cells were the most upstream of the identified apoptosis-related events studied here. These effects became prominent between 2 and 4 h p.i. (Fig. and ). Remarkably, switching on of the apoptotic program in a significant proportion of infected cells could be detected as early as 1.5 to 2 h p.i. (4
). The upstream reactions occurring at the very early stages of infection prior to the mitochondrial damage have yet to be elucidated. Since several poliovirus proteins, i.e., 2A (26
), 3C (6
), and possibly some others, exhibit apoptosis-inducing activity, more than one mechanism of the poliovirus-triggered mitochondrial damage may conceivably be involved. Theoretically, the virus-activated pathway leading to cytochrome c
detachment from cardiolipin and its subsequent efflux into the cytoplasm through an altered outer mitochondrial membrane (2
) may include activation of some proapoptotic members of the Bcl-2 family of proteins, such as Bid (1
); direct interaction of the viral protein and mitochondria (22
); some other indirect effects; or a combination of these factors. Processing of Bid was indeed detected in abortively infected HeLa cells destined for apoptosis. However, since permeabilization of the outer mitochondrial membrane occurred upon productive infection as well, when no appreciable Bid processing was observed, we concluded that Bid was not the primary inducer of mitochondrial damage. More likely, processing of Bid was accomplished by an already activated effector caspase, e.g., caspase-3 (61
), and represented a component of the loop involved in the amplification of the apoptotic signal. Caspase-2, which was recently demonstrated to be a key component in the mitochondrial-damage pathway activated by certain apoptotic inducers (26a
), does not appear to play such a role in MCF-7 cells (37a
) and therefore could hardly be responsible for the poliovirus-triggered apoptosis in the derivatives of these cells.
It is perhaps appropriate to note that alterations in the oxidative functions of mitochondria have been reported to occur early in poliovirus infection (36
Although they were not studied here, mitochondrial alterations may also be accompanied by the release of other components of the apoptotic machinery, such as the mitochondrial stores of some procaspases (e.g., procaspase-9 and procaspase-3) (43
) and the Smac/Diablo protein acting as an inhibitor of the cellular antiapoptotic IAP proteins (18
), as well as proteins capable of killing cells in a caspase-independent way, such as the apoptosis-inducing factor AIF (59
), endonuclease G (39
), and Omi/HtrA2 serine protease (28
). The fact that in the absence of caspase-9 and caspase-3 the virus-triggered apoptotic response was strongly suppressed indicated that mechanisms independent of these caspases (e.g., AIF mediated), if involved, hardly played a decisive role in this response.
It may be noted that apoptosis induced by proteins of some other viruses, e.g., human immunodeficiency virus (32
) or chicken anemia virus (16
), may also involve the mitochondrion-dependent pathway.
Once cytochrome c
is released, the energy-dependent generation of high-molecular-mass complexes, the apoptosomes, also involving Apaf-1 and procaspase-9, is expected to occur, ensuring limited autoproteolysis of the latter and the appearance of active caspase-9 (40
). In poliovirus-infected cells destined for apoptosis, the proper procaspase-9 cleavage was documented here by Western blotting, and the key role of caspase-9 in the poliovirus-triggered apoptotic pathway was demonstrated by a dramatic suppression of the development of apoptosis-related nuclear damage in cells lacking appropriate activity (i.e., MCF-Cas3/C9DN).
The next downstream essential component of the poliovirus-induced apoptotic pathway is caspase-3. Previously, activation of Asp-Glu-Val-Asp (DEVD)-specific caspases (4
) and cleavage of procaspase-3 (42
) have been observed in poliovirus-infected cells undergoing apoptosis. As shown here, the lack of caspase-3 in MCF-Cas3cs cells resulted in the nearly complete absence of key signs of apoptosis. Thus, caspase-3 is an essential component of the poliovirus-triggered apoptotic pathway, while other known effector caspases, e.g., caspase-6 and caspase-7, appear to be unable to efficiently replace caspase-3 in this process.
One may speculate that the activation of caspase-3 in abortively infected cells might be accomplished not only by the mitochondrion-dependent pathway but also by some other mechanisms. For example, the involvement of caspase-12 known to be activated in response to ER stress (45
) cannot be ruled out, since drastic ER alterations accompany poliovirus infection (56
). As shown recently, this enzyme is able to activate caspase-9 through a mitochondrion-independent mechanism (44a
). The ER-dependent pathway was reported to be activated, for example, upon infection with respiratory syncytial virus (8
). Although the potential role of this and some other mechanisms in poliovirus-induced apoptosis has yet to be explored, the marked antiapoptotic activity of Bcl-2 argues for a mitochondrion-mediated mode. Thus, our results suggest that the major poliovirus-triggered apoptotic pathway in the cells studied here includes consecutive activation of caspase-9 and caspase-3 and appears to be caused by cytochrome c
release (Fig. ).
FIG. 7. Schematic representation of the major poliovirus-triggered apoptotic pathway as characterized in the present study. Poliovirus infection results in mitochondrial damage and efflux of cytochrome c (cyt c) into the cytoplasm. Together with Apaf-1 and cytochrome (more ...)
Concurrently with turning on the apoptotic program and despite the potential availability of all of the necessary components of the major apoptotic pathway, the overwhelming majority of productively infected HeLa and MCF-Cas3 cells develop CPE rather than typical apoptosis. This is due to the antiapoptotic function(s) of poliovirus capable of interrupting implementation of the ongoing apoptotic program (4
). Since cytochrome c
release proceeds upon productive infection essentially as efficiently as upon abortive infection, we conclude that the major target of the putative viral antiapoptotic function(s) is downstream of the mitochondrial damage. In this respect, the antiapoptotic functions of poliovirus differ from those of several other viruses, e.g., some herpesviruses and adenoviruses, which encode proteins able to stabilize mitochondria and thus prevent the release of cytochrome c
The molecular basis of poliovirus interference with the activation of caspase-9 is still unclear. The appearance of caspase-9-related proteins with unexpected electrophoretic mobility in productively infected cells may suggest that the viral antiapoptotic function involves aberrant processing, e.g., alternative proteolysis, of procaspase-9. The amino acid sequence of human procaspase-9 contains a YG dipeptide, a canonical cleavage site for the poliovirus 2A protease (50
). The putative cleavage should generate peptides of 251 and 165 amino acid residues, respectively. Neither of them could be related to the observed aberrant polypeptide bands. Since the specificity of poliovirus 2A protease, especially its trans
-acting activity, is highly dependent on the context of the cleavage site (29
), it is not surprising that the YG bond in procaspase-9 does not appear to be the target for this protease. No canonical cleavage site (QG [50
]) for the poliovirus protease 3C is present in procaspase-9. Thus, either a viral protease cleaves the proenzyme at suboptimal sites or an infection-stimulated cellular proteolytic activity is involved. Interestingly, a Ca-dependent protease, calpain, is known to degrade and inactivate procaspase-9, as well as some other caspases (15
). In this regard, it may be noted that a misbalance of ions (Ca2+
included) caused by alterations in the permeability of cellular membranes in poliovirus-infected cells is a well-documented phenomenon (13
If the aberrant cleavage of procaspase-9 occurs closer to its N terminus, it may be accompanied by the removal or inactivation of the caspase recruitment domain (CARD), preventing recruitment of the proenzyme into the apoptosome (72
). It may be appropriate to note that the liberated CARD itself may perform an antiapoptotic function by activating NF-κB (58
The identification of aberrant processing of procaspase-9 as a putative component of the poliovirus antiapoptotic activity does not necessarily rule out the existence of additional relevant mechanisms. A variety of factors are known to impair the recruitment of caspase-9 into apoptosomes or its subsequent activation (7
), and they may be regarded as potential targets for viral antiapoptotic functions.
Although the major apoptotic pathway operating in poliovirus-infected cells is mitochondrion mediated, other pathways may potentially be involved under some settings. As already mentioned, the ER stress-mediated pathway also potentially contributes to the cellular response to poliovirus infection. The infection may modulate the extrinsic, receptor-mediated apoptotic pathway as well. For example, expression of poliovirus protein 2A renders the cells more sensitive to the apoptosis-inducing effect of TNF (47
). On the other hand, viral infection and, in particular, expression of the viral protein 3A results in depletion of TNF receptor (47
) and receptors for some other cytokines (47a
) from the plasma membrane, decreasing the sensitivity of the cells to these cytokines.
Thus, although we did not study it, the fate of the infected cell depends on a complex network of interacting cellular and viral proteins and is host specific as well as virus specific. The results reported here contribute to a deeper understanding of this network. The type of death of infected cells is a significant factor in the pathogenesis of viral diseases. CPE, but not apoptosis, is generally accompanied by an inflammatory reaction, and the presence or absence of this reaction should markedly affect the clinical patterns of viral diseases.