**2.3.2 Glycogen synthase kinase-3β**

In addition to the roles of caspases during cell death, the actions of glycogen synthase kinase-3 β (GSK-3β) have also been implicated in the induction of cytopathic effect by CVB3 replication (Yuan et al., 2005; Zhang et al., 2003). Phosphorylation of transcription factors by GSK-3β can lead to either the activation or down-regulation of transcription, and activation of GSK-3β has been observed relatively early in the CVB3 replication cycle (Yuan et al. 2005). The catenins are poly-phosphorylated by activated GSK-3β, leading to degradation in the proteasome (Doble and Woodgett, 2003; Harwood, 2001). During CVB3 infection, the activation of GSK-3β has been proposed to lead to instability of cell viability due to

effect.

**2.3.5 Necroptosis?** 

insight.

Cellular and Immunological Regulation of Viral Myocarditis 275

whereas CVB3 will have induced apoptosis and destroyed a similar culture of HeLa cells in just 8 – 10 hours. The rapid nature of CVB3 induced cell lysis suggests the synergy between numerous death pathways 'criss-cross' and interact to enhance their collective cell-death

The loss of checked and contained cell death results in necrosis and lysis of the CVB3 infected host cell. Even though the pathways of cell death are clearly defined in this arena, the precise mechanisms of initiation are still unclear, and potential pathway interactions are unknown. Many reports have indicated that a particular pathway may be involved in CVB3 induced cell death although there hasn't been 100 % recovery of cell viability demonstrated from experiments that inhibit just one pathway. However, zVAD, a pan caspase inhibitor does not completely inhibit virus induced death and lysis just as GSK3β inhibition doesn't completely inhibit cell-rounding and death. Therefore there is still much to be learned about the initiation and interaction of cell death pathways during CVB3 infection, but we may never be able to completely inhibit CVB3 induced cell death because once virus replication

Necroptosis (necrotic apoptosis) is a tumour necrosis factor receptor activated form of programmed cell death that is activated by the same class of receptors that activate caspase-8 mediated apoptosis (Declercq et al., 2009; Hitomi et al., 2008; Yuan and Kroemer, 2010). This is a more recently discovered pathway of cell death that has not been reported in the context of CVB3 infection. However, instead of caspase-8 activation that normally results from TNF receptor ligation there is activation of a receptor interacting protein (RIP)-1/RIP-3 pathway that results in rupture of the cell through loss of membrane integrity; a mode of cell death reminiscent of CVB3 induced cell lysis. Transgenic caspase-8 null animals undergo RIP-3 necroptosis instead of apoptosis, leading to embryonic lethality and nonviability of the line. However caspase-8 -/-, RIP-3 -/- double transgenic mice are viable, suggesting that caspase- 8 holds RIP-3 'in check', preventing initiation of programmed necrosis (Kaiser et al., 2011). The necroptosis pathway has not yet been described with respect to CVB3 infection but given the similarities between CVB3 induced cell lysis and necroptosis this pathway of cell death and the RIP-1/RIP-3 proteins may provide some

There have been reports showing that cell death activated early in the virus life cycle significantly inhibits viral replication (Zhang et al., 2010). Release of the cell contents prior to progeny virion production and more programmed processes of cell death will halt virus replication by killing the cell prior to assembly of progeny virus virion or confinement of

Virus replication is cytotoxic and can cause cell death before production of progeny virion, terminating the life-cycle of the virus. For example, reactive oxygen species are a byproduct of CVB3 replication very early in the life-cycle of the virus, placing stress on the cell such that the mitochondrial cytochrome *c* pathway of caspase-9 mediated cell death might be activated prior to virion assembly. To prevent premature death of the host cell coxsackievirus triggers the activation of pro-survival proteins and their associated cascades to suppress cell lysis until progeny virion can be assembled. Perhaps the most studied pro-

is initiated the cell may already be past the threshold of viability.

**2.4 Virus induced cell death and lysis – 'Timing is everything'** 

progeny virion into vimentin cages, respectively.

degradation of a protein called β-catenin (Figure 1C). The catenins interact with the actin cytoskeleton and act to maintain morphological integrity by connecting the actin cytoskeleton with the cell membrane. The loss of these connections through enhanced GSK-3β activation and β-catenin degradation is consistent with the profound cell rounding that occurs during intermediate to late phases of CVB3 replication. The accumulation of βcatenin in the cytoplasm leads to translocation into the nucleus where it transactivates survival signalling via T-cell factor (TCF)/lymphocyte enhancer factor (LEF) family members (Doble and Woodgett, 2003; Hardt and Sadoshima, 2002; Harwood, 2001). Consistent with these findings, the inhibition of GSK-3β or the over-expression of β-catenin resulted in the suppression of viral progeny release, but not viral protein production (Yuan et al., 2005). Therefore GSK-3β is pivotal in regulating the rupture of infected cells for progeny release.

#### **2.3.3 The endoplasmic reticulum unfolded protein response**

CVB3 induces the unfolded protein response (UPR), which is an acute stress response that leads to ER stress (Zhang et al., 2010). Poliovirus, CVB3 and other viruses have been shown to replicate their genomes and translate protein on the surfaces of double membrane vesicles derived from the ER and autophagosomes (Suhy et al., 2000; Wong et al., 2008; Zhang et al., 2011). This activity in combination with interruption of regular protein translation and a massive upregulation of protein production by the virus probably leads to activation of the UPR. This process involves 3 different pathways of independent activation, initiated by 3 respective stress sensors in the proximal ER: ATF6a, IRE1-XBP-1, (X box binding protein 1), and PERK (PKR-like ER protein kinase). It has been shown that infection with CVB3 invokes activation of ATF6a and XBP-1, possibly through replication of the virus on the surface of double membrane vesicles derived from the ER and autophagosome (Wong et al., 2008). Other viruses that replicate in the cytosol, such as West Nile Virus, have been shown to activate this pathway to promote virus replication and cell death ((Medigeshi et al., 2007) review). The UPR response to massive viral protein synthesis and usurped cellular pathways most likely acts to initiate or augment the onset of death in the infected host cell. However if these pathways are activated too early in the virus replication cycle the cell will die before virus protein production and virus progeny can be completed and released, respectively. This would surely result in the termination of virus replication. Zhang et al. showed that expression of the UPR response protein, XBP-1 acts to delay the onset of cell lysis and release of progeny virion (Figure 1B). Therefore, pathways like XBP-1 are used by the virus to counter early destruction of the cell; so CVB3 has evolved mechanisms to prevent premature death of the infected cell. Major cellular survival pathways, like Akt, are also invoked to counter the death reflex, which we will discuss later in the chapter.

#### **2.3.4 Cell death and lysis through activation of many simultaneous pathways**

The overwhelming and rapid activation of many different pathways simultaneously leads to cell death and lysis (Figure 1), liberating progeny virion. It is possible, that cell death pathways may even be synergistic, additively driving the cell toward more rapid and thus uncontrolled death, leading to lysis. The cytochrome c- caspase-9 pathway requires a minimum of 15 hours to kill a cell that has been chemically induced, almost twice as long as it takes CVB3 to lyse an infected cell (Qu and Qing, 2004). The plant toxin abrin requires upwards of 15 – 36 hours to kill a culture of HeLa cells (Qu and Qing, 2004), the E2 protein of human papillomavirus requires 20 – 24 hrs to induce apoptosis (Desaintes et al., 1999), whereas CVB3 will have induced apoptosis and destroyed a similar culture of HeLa cells in just 8 – 10 hours. The rapid nature of CVB3 induced cell lysis suggests the synergy between numerous death pathways 'criss-cross' and interact to enhance their collective cell-death effect.

The loss of checked and contained cell death results in necrosis and lysis of the CVB3 infected host cell. Even though the pathways of cell death are clearly defined in this arena, the precise mechanisms of initiation are still unclear, and potential pathway interactions are unknown. Many reports have indicated that a particular pathway may be involved in CVB3 induced cell death although there hasn't been 100 % recovery of cell viability demonstrated from experiments that inhibit just one pathway. However, zVAD, a pan caspase inhibitor does not completely inhibit virus induced death and lysis just as GSK3β inhibition doesn't completely inhibit cell-rounding and death. Therefore there is still much to be learned about the initiation and interaction of cell death pathways during CVB3 infection, but we may never be able to completely inhibit CVB3 induced cell death because once virus replication is initiated the cell may already be past the threshold of viability.

### **2.3.5 Necroptosis?**

274 Myocarditis

degradation of a protein called β-catenin (Figure 1C). The catenins interact with the actin cytoskeleton and act to maintain morphological integrity by connecting the actin cytoskeleton with the cell membrane. The loss of these connections through enhanced GSK-3β activation and β-catenin degradation is consistent with the profound cell rounding that occurs during intermediate to late phases of CVB3 replication. The accumulation of βcatenin in the cytoplasm leads to translocation into the nucleus where it transactivates survival signalling via T-cell factor (TCF)/lymphocyte enhancer factor (LEF) family members (Doble and Woodgett, 2003; Hardt and Sadoshima, 2002; Harwood, 2001). Consistent with these findings, the inhibition of GSK-3β or the over-expression of β-catenin resulted in the suppression of viral progeny release, but not viral protein production (Yuan et al., 2005). Therefore GSK-3β is pivotal in regulating the rupture of infected cells for

CVB3 induces the unfolded protein response (UPR), which is an acute stress response that leads to ER stress (Zhang et al., 2010). Poliovirus, CVB3 and other viruses have been shown to replicate their genomes and translate protein on the surfaces of double membrane vesicles derived from the ER and autophagosomes (Suhy et al., 2000; Wong et al., 2008; Zhang et al., 2011). This activity in combination with interruption of regular protein translation and a massive upregulation of protein production by the virus probably leads to activation of the UPR. This process involves 3 different pathways of independent activation, initiated by 3 respective stress sensors in the proximal ER: ATF6a, IRE1-XBP-1, (X box binding protein 1), and PERK (PKR-like ER protein kinase). It has been shown that infection with CVB3 invokes activation of ATF6a and XBP-1, possibly through replication of the virus on the surface of double membrane vesicles derived from the ER and autophagosome (Wong et al., 2008). Other viruses that replicate in the cytosol, such as West Nile Virus, have been shown to activate this pathway to promote virus replication and cell death ((Medigeshi et al., 2007) review). The UPR response to massive viral protein synthesis and usurped cellular pathways most likely acts to initiate or augment the onset of death in the infected host cell. However if these pathways are activated too early in the virus replication cycle the cell will die before virus protein production and virus progeny can be completed and released, respectively. This would surely result in the termination of virus replication. Zhang et al. showed that expression of the UPR response protein, XBP-1 acts to delay the onset of cell lysis and release of progeny virion (Figure 1B). Therefore, pathways like XBP-1 are used by the virus to counter early destruction of the cell; so CVB3 has evolved mechanisms to prevent premature death of the infected cell. Major cellular survival pathways, like Akt, are

also invoked to counter the death reflex, which we will discuss later in the chapter.

**2.3.4 Cell death and lysis through activation of many simultaneous pathways** 

The overwhelming and rapid activation of many different pathways simultaneously leads to cell death and lysis (Figure 1), liberating progeny virion. It is possible, that cell death pathways may even be synergistic, additively driving the cell toward more rapid and thus uncontrolled death, leading to lysis. The cytochrome c- caspase-9 pathway requires a minimum of 15 hours to kill a cell that has been chemically induced, almost twice as long as it takes CVB3 to lyse an infected cell (Qu and Qing, 2004). The plant toxin abrin requires upwards of 15 – 36 hours to kill a culture of HeLa cells (Qu and Qing, 2004), the E2 protein of human papillomavirus requires 20 – 24 hrs to induce apoptosis (Desaintes et al., 1999),

**2.3.3 The endoplasmic reticulum unfolded protein response** 

progeny release.

Necroptosis (necrotic apoptosis) is a tumour necrosis factor receptor activated form of programmed cell death that is activated by the same class of receptors that activate caspase-8 mediated apoptosis (Declercq et al., 2009; Hitomi et al., 2008; Yuan and Kroemer, 2010). This is a more recently discovered pathway of cell death that has not been reported in the context of CVB3 infection. However, instead of caspase-8 activation that normally results from TNF receptor ligation there is activation of a receptor interacting protein (RIP)-1/RIP-3 pathway that results in rupture of the cell through loss of membrane integrity; a mode of cell death reminiscent of CVB3 induced cell lysis. Transgenic caspase-8 null animals undergo RIP-3 necroptosis instead of apoptosis, leading to embryonic lethality and nonviability of the line. However caspase-8 -/-, RIP-3 -/- double transgenic mice are viable, suggesting that caspase- 8 holds RIP-3 'in check', preventing initiation of programmed necrosis (Kaiser et al., 2011). The necroptosis pathway has not yet been described with respect to CVB3 infection but given the similarities between CVB3 induced cell lysis and necroptosis this pathway of cell death and the RIP-1/RIP-3 proteins may provide some insight.

#### **2.4 Virus induced cell death and lysis – 'Timing is everything'**

There have been reports showing that cell death activated early in the virus life cycle significantly inhibits viral replication (Zhang et al., 2010). Release of the cell contents prior to progeny virion production and more programmed processes of cell death will halt virus replication by killing the cell prior to assembly of progeny virus virion or confinement of progeny virion into vimentin cages, respectively.

Virus replication is cytotoxic and can cause cell death before production of progeny virion, terminating the life-cycle of the virus. For example, reactive oxygen species are a byproduct of CVB3 replication very early in the life-cycle of the virus, placing stress on the cell such that the mitochondrial cytochrome *c* pathway of caspase-9 mediated cell death might be activated prior to virion assembly. To prevent premature death of the host cell coxsackievirus triggers the activation of pro-survival proteins and their associated cascades to suppress cell lysis until progeny virion can be assembled. Perhaps the most studied pro-

Cellular and Immunological Regulation of Viral Myocarditis 277

Virus replication

**B**

PERK

Fig. 1. Pathways of cell death in CVB3 infected cardiomyocytes. A. Coxsackievirus replication results in production of reactive oxygen species which promote the release of cytochrome c from mitochondria. The formation of Bak and Bax at the membrane surface mediate passage of cytochrome c into the cytosol. Overexpression of the anti-apoptotic proteins BCl 2 and BCl-XL during CVB3 infection significantly reduce cytochrome *c* release and activation of the caspase-9 pathway of apoptosis, which leads to caspase-3 death effector caspase activation. ROS = reactive oxygen species. B. The unfolded protein response

(UPR) of the endoplasmic reticulum (ER) is activated during CVB3 infection of

during CVB3 infection resulting in phosphorylation and subsequent proteosomal degradation of its target, β –catenin, in the cytosol. C. Loss of β –catenin in the cytosol results in loss of connections between the actin cytoskeleton and cadherin receptors, via connections to α and β –catenins. D. Less β –catenin in the cytosol means that there will be less available to translocate across the nucleus to activate survival gene expression via TCF

cardiomyocytes leading to activation of all 3 known pathways of the UPR: spliced XBP-1s, PERK and ATF6a ER stress sensors are all activated during CVB3 infection. Virus replication occurs on the surface of double membrane vesicles and membrane derived from the ER, activating the ER stress sensors. The result of XBP-1s activation is to slow the onset of cell death to prevent the cell from rupturing prior to the assembly of progeny virus virions. UPR = unfolded protein response. C and D. Glycogen synthase kinase -3β (GSK-3β) is activated

UPR

XBP-1s

ROS

**A**

ATF-6a

**TCF TBP**

**C**

**D**

**Bcl-XL**

**Casp 3**

and TBP transcription factors.

Programmed cell death – endonuclease activation, DNA fragmentation and PARP cleavage

**C**

**Bcl-2**

**C C C**

**Casp 9**

P P P

GSK-3β

Cytoskeleton/structural integrity

Survival signalling

survival protein, Akt, is activated during the CVB3 replication cycle (Esfandiarei et al., 2007; Esfandiarei et al., 2004; Liu et al., 2008). It may appear counter-intuitive for a cytolytic virus to evolve to activate pro-survival pathways, particularly if the virus requires the cell to lyse at the end of the cycle. However, as we discussed in section 2.1 CVB3 replication is cytotoxic from the onset so pro-survival pathways may be selected evolutionarily to support the survival of the cell while viral protein synthesis and viral progeny assembly are taking place. These mechanisms of cell survival during virus infection act in favour of viral replication because they allow maximum amounts of virus progeny to be assembled prior to cell lysis and release of viral progeny. Subsequently, these same survival pathways must be inhibited to then permit cell death and lysis for release of assembled viral progeny virions.

#### **2.5 Coxsackievirus induced cell death: many dimensions of complexity**

It is now apparent that several cell death pathways and perhaps dozens of proteins are activated in a web of molecular interaction in order to short circuit normal death mechanisms, to cause rapid and aberrant rupture of the infected cell (Figure 1). New pathways of cell death, such as necroptosis, may as yet be shown to mediate death of the CVB3 infected cell. Nevertheless, there already exists a large web of seemingly disparate pathways of CVB3 induced cellular lysis. As we discussed earlier, it is difficult to determine the foundation pathway that is responsible for virally induced cell lysis of infected cardiomyocytes, and which ones are triggered, merely as a matter of coincident activation. New methods of data organisation and analysis are clearly needed as more pathways and mechanisms of CVB3 induced cell death are revealed. Knowledge of the truly pertinent and central pathways of cell death could lead to the most robust antivirals. Toward the end of this chapter we will discuss the use of systems biology to organise the myriad of pathways activated during CVB3 infection (proteolytic and kinase) and how it could be applied to elucidate the dominant pathways of virus induced cell death.
