**2.1 The cytotoxic nature of enterovirus replication**

Coxsackievirus B3 is a member of the *Enteroviridae* of the *Picornaviridae* family, which are fast replicating and exquisitely cytotoxic viruses. Another member of this family is poliovirus, which is also highly cytotoxic, producing a paralytic syndrome reminiscent of the aseptic meningitis and encephalitis sometimes caused by CVB3. The very nature of enterovirus replication makes these viruses very cytotoxic because the infected cell must burst to permit the release of progeny virus virions for infection of bystander cells and further replication cycles. This aspect of CVB3 replication means that every virus infected cell will die within 8 – 24 hrs of infection, releasing numerous rounds of progeny virus into the heart before the innate immune system has had a chance to control infection. The degree of immune infiltration and damage inflicted on the heart muscle is therefore a function of the amount of virus inoculum that takes hold in the heart, dictating the degree of damage done to the myocardium.

The virus polarises the protein expression machinery entirely to the benefit of the virus by a number of mechanisms and so in combination with the inability of the cell to perform its natural housekeeping functions inevitably leads to cell destruction. The viral proteases act to cleave capped cellular mRNAs thereby skewing protein translation in the cell from capdependent translation initiation to internal ribosome entry site (IRES) translation. Coxsackievirus B3 translates it's RNA genome via IRES translation initiation so destruction of the cell's own capped mRNAs removes any competition of the virus' own IRES RNAs for the cell's ribosomes. This usurping of control of the cell's protein expression machinery effectively brings the normal housekeeping function of the cell to a halt.

The massive amount of protein produced by the virus, devolution of the endoplasmic reticulum (ER) and other membranous structures combined with the oxidative stress placed upon the cell by virus replication, all destabilise the homeostasis of the infected cell. The large protein-nucleic acid aggregates that make up progeny virion are not normal to the cell and induce numerous UPR responses; due to their toxic nature, protein aggregates are

death.

(Figure 1A).

by the virus to drive cell death.

**2.3.2 Glycogen synthase kinase-3β**

Cellular and Immunological Regulation of Viral Myocarditis 273

Horvitz, 1990) reviewed in (Yuan and Horvitz, 2004)]. The first cellular pathways implicated in cell death due to CVB3 infection were also caspase mediated (Carthy et al., 1998; Colston et al., 1998), since these were the predominant mechanisms of cell death being studied at the time (Andrade et al., 1998; Atkinson et al., 1998; Barry et al., 2000). As new mechanisms of cell death were discovered, such as GSK-3β, some were attributed to virus induced cell

Comprehensive studies of caspase cleavage and activation during infection have demonstrated a pan-caspase activation profile during viral infection (Carthy et al., 1998; Carthy et al., 2003), from activation of the tumour necrosis factor (TNF) receptor activated caspase-8 to the mitochondrial cytochrome *c* release that results in the activation of caspase-9. All of the above mentioned pathways converge upon the activation of the effector caspase, caspase-3. The most notable caspase-8 activated pathway is triggered by Fas ligand binding and activation of Fas receptor, a member of the TNF superfamily of receptors. Various oxidative states, toxins and aberrant protein expression can lead to a permeable mitochondrial membrane that releases cytochrome *c* into the cytoplasm which leads to caspase-9 activation. Though the caspase-8 and -9 pathways are quite different in that they are extrinsic and intrinsic pathways of cell death, respectively, they both lead to cleavage and activation of caspase-3, a death effector caspase. In cells destined to die by either of these routes of apoptosis there is cleavage of poly ADP-ribose polymerase (PARP) and DNA fragmentation factor (DFF) by caspase-3. Cleavage of DFF by this caspase reveals an endonuclease that enters the nucleus and cleaves DNA, resulting in DNA fragmentation

Two of the experimental hallmarks of apoptotic cell death are PARP fragmentation, as observed on a Western blot, and fragmented genomic DNA as viewed by agarose gel electrophoresis. It was demonstrated that inhibition of caspase-3 PARP and DFF cleavage did not inhibit virus induced cell death completely (Carthy et al., 1998), however some alleviation of cytopathic effect was notable. The broad spectrum zVAD.fmk caspase inhibitor is not 100 % effective at inhibiting all caspases, and there is the possibility that in the absence of caspase activation that a redundant mechanism of cell death becomes dominant in mediating cytotoxicity. In this scenario we would expect zVAD.fmk to appear ineffectual at inhibiting cell death. It was apparent that the caspases were activated though this was clearly not the mechanism that was entirely responsible for CVB3 induced cytopathic effect. Below we will outline more mechanisms responsible for cell death and that may run parallel to caspase mediated cytotoxicity. Later in this chapter we will also cover new systems biology approaches that could be applied toward the understanding of the dominant/ pertinent pathways of CVB3 induced cell lysis and the networks employed

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

normally sequestered to vimentin-cytoskeleton encased aggrosomes (Garcia-Mata et al., 1999; Johnston et al., 1998). For example, the irregular folding of viral proteins can lead to sequestering of virus protein products into cellular aggresomes (Spiropoulou et al., 2003). These combined facets of CVB3 replication contribute to the inherently toxic nature of virus replication, even in the absence of the onset of programmed cell death.

### **2.2 Virus entry into the host cell**

The replication cycle of CVB3 in the cell begins in a very similar manner to poliovirus infection via receptor mediated endocytosis into the target host cell. Coxsackievirus B3 binds its cellular receptor(s), the coxsackie-adenovirus receptor (CAR) and ,sometimes, decay acceleration factor (DAF), to achieve entry via endocytosis into the host cell. Once the virus has achieved delivery inside the cell there is massive rearrangement of internal membrane structures and then complete usurping of the host cell protein translation machinery for the purposes of virus replication and virion assembly. The RNA polarity (positive-polarity RNA) of the CVB3 genome means that viral polypeptide can be translated directly after delivery into the host cell. All of the proteins encoded by the CVB3 genome are expressed as 1 poly-protein that is then cleaved by virus proteases, 2A and 3C, into the individual viral proteins VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, and 3D.

#### **2.3 Death and lysis of virus infected cells**

Direct virus-induced damage inflicted by CVB3 upon the infected cell is through simultaneous activation of multiple cell death pathways, by direct and indirect mechanisms. For example, all of the known caspase cell death activation pathways are activated during CVB3 infection: the FAS/FADD receptor-mediated caspase- 8 pathway and the mitochondrial cytochrome c -caspase- 9 pathway are activated to ultimately trigger the death effector, caspase-3 (Carthy et al., 1998; Carthy et al., 2003). Normally, the activation of caspases during apoptosis leads to programmed cell death: cleavage of cellular DNA by endonucleases and shrinking of the cell's contents into a cytoskeleton-vimentin cage, for engulfment by phagocytes at some later time. This process is programmed, and most importantly, 'neat,' preventing the dissemination of the dead cell's contents throughout the tissue. The debris released from cells are hydrolytic and oxidative, some are apoptotic, which would lead to bystander necrosis of surrounding cells and tissue if not disposed of 'neatly' through programmed cell death. However, CVB3 infected cells do not die in a programmed and neat manner. The act of virus replication; viral proteases and activation of numerous cell death pathways all at once cause infected cells to rapidly lyse and release cellular contents, along with virus progeny virions, for further rounds of replication in surrounding bystander cells.

Necrotic cell death during CVB3 infection is most likely caused by a myriad of pathways that act in concert to result in the uncontrolled lysis of the cell for the purpose of virus progeny release. Evolution of the virus has favoured a more chaotic lysis/necrosis type death as opposed to a programmed and controlled method of killing the host cell. We will summarise the pathways of CVB3 induced cell death below:

#### **2.3.1 Caspase-induced cell death during CVB3 infection**

The caspase pathways of apoptosis were the first mechanisms of programmed cell death that were discovered [(Lazebnik et al., 1994; Miura et al., 1993; Yuan et al., 1993; Yuan and

normally sequestered to vimentin-cytoskeleton encased aggrosomes (Garcia-Mata et al., 1999; Johnston et al., 1998). For example, the irregular folding of viral proteins can lead to sequestering of virus protein products into cellular aggresomes (Spiropoulou et al., 2003). These combined facets of CVB3 replication contribute to the inherently toxic nature of virus

The replication cycle of CVB3 in the cell begins in a very similar manner to poliovirus infection via receptor mediated endocytosis into the target host cell. Coxsackievirus B3 binds its cellular receptor(s), the coxsackie-adenovirus receptor (CAR) and ,sometimes, decay acceleration factor (DAF), to achieve entry via endocytosis into the host cell. Once the virus has achieved delivery inside the cell there is massive rearrangement of internal membrane structures and then complete usurping of the host cell protein translation machinery for the purposes of virus replication and virion assembly. The RNA polarity (positive-polarity RNA) of the CVB3 genome means that viral polypeptide can be translated directly after delivery into the host cell. All of the proteins encoded by the CVB3 genome are expressed as 1 poly-protein that is then cleaved by virus proteases, 2A and 3C, into the

Direct virus-induced damage inflicted by CVB3 upon the infected cell is through simultaneous activation of multiple cell death pathways, by direct and indirect mechanisms. For example, all of the known caspase cell death activation pathways are activated during CVB3 infection: the FAS/FADD receptor-mediated caspase- 8 pathway and the mitochondrial cytochrome c -caspase- 9 pathway are activated to ultimately trigger the death effector, caspase-3 (Carthy et al., 1998; Carthy et al., 2003). Normally, the activation of caspases during apoptosis leads to programmed cell death: cleavage of cellular DNA by endonucleases and shrinking of the cell's contents into a cytoskeleton-vimentin cage, for engulfment by phagocytes at some later time. This process is programmed, and most importantly, 'neat,' preventing the dissemination of the dead cell's contents throughout the tissue. The debris released from cells are hydrolytic and oxidative, some are apoptotic, which would lead to bystander necrosis of surrounding cells and tissue if not disposed of 'neatly' through programmed cell death. However, CVB3 infected cells do not die in a programmed and neat manner. The act of virus replication; viral proteases and activation of numerous cell death pathways all at once cause infected cells to rapidly lyse and release cellular contents, along with virus progeny virions, for further rounds of replication in

Necrotic cell death during CVB3 infection is most likely caused by a myriad of pathways that act in concert to result in the uncontrolled lysis of the cell for the purpose of virus progeny release. Evolution of the virus has favoured a more chaotic lysis/necrosis type death as opposed to a programmed and controlled method of killing the host cell. We will

The caspase pathways of apoptosis were the first mechanisms of programmed cell death that were discovered [(Lazebnik et al., 1994; Miura et al., 1993; Yuan et al., 1993; Yuan and

summarise the pathways of CVB3 induced cell death below:

**2.3.1 Caspase-induced cell death during CVB3 infection** 

replication, even in the absence of the onset of programmed cell death.

individual viral proteins VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, and 3D.

**2.2 Virus entry into the host cell** 

**2.3 Death and lysis of virus infected cells** 

surrounding bystander cells.

Horvitz, 1990) reviewed in (Yuan and Horvitz, 2004)]. The first cellular pathways implicated in cell death due to CVB3 infection were also caspase mediated (Carthy et al., 1998; Colston et al., 1998), since these were the predominant mechanisms of cell death being studied at the time (Andrade et al., 1998; Atkinson et al., 1998; Barry et al., 2000). As new mechanisms of cell death were discovered, such as GSK-3β, some were attributed to virus induced cell death.

Comprehensive studies of caspase cleavage and activation during infection have demonstrated a pan-caspase activation profile during viral infection (Carthy et al., 1998; Carthy et al., 2003), from activation of the tumour necrosis factor (TNF) receptor activated caspase-8 to the mitochondrial cytochrome *c* release that results in the activation of caspase-9. All of the above mentioned pathways converge upon the activation of the effector caspase, caspase-3. The most notable caspase-8 activated pathway is triggered by Fas ligand binding and activation of Fas receptor, a member of the TNF superfamily of receptors. Various oxidative states, toxins and aberrant protein expression can lead to a permeable mitochondrial membrane that releases cytochrome *c* into the cytoplasm which leads to caspase-9 activation. Though the caspase-8 and -9 pathways are quite different in that they are extrinsic and intrinsic pathways of cell death, respectively, they both lead to cleavage and activation of caspase-3, a death effector caspase. In cells destined to die by either of these routes of apoptosis there is cleavage of poly ADP-ribose polymerase (PARP) and DNA fragmentation factor (DFF) by caspase-3. Cleavage of DFF by this caspase reveals an endonuclease that enters the nucleus and cleaves DNA, resulting in DNA fragmentation (Figure 1A).

Two of the experimental hallmarks of apoptotic cell death are PARP fragmentation, as observed on a Western blot, and fragmented genomic DNA as viewed by agarose gel electrophoresis. It was demonstrated that inhibition of caspase-3 PARP and DFF cleavage did not inhibit virus induced cell death completely (Carthy et al., 1998), however some alleviation of cytopathic effect was notable. The broad spectrum zVAD.fmk caspase inhibitor is not 100 % effective at inhibiting all caspases, and there is the possibility that in the absence of caspase activation that a redundant mechanism of cell death becomes dominant in mediating cytotoxicity. In this scenario we would expect zVAD.fmk to appear ineffectual at inhibiting cell death. It was apparent that the caspases were activated though this was clearly not the mechanism that was entirely responsible for CVB3 induced cytopathic effect. Below we will outline more mechanisms responsible for cell death and that may run parallel to caspase mediated cytotoxicity. Later in this chapter we will also cover new systems biology approaches that could be applied toward the understanding of the dominant/ pertinent pathways of CVB3 induced cell lysis and the networks employed by the virus to drive cell death.
