**4.1 Defining cell signalling network models in virus-host interactions**

Host cells have evolved complex systems to detect and eradicate viruses; on the other hand, viruses have evolved mechanisms to compromise essential cellular processes and suppress the host cell defence. In these complex systems, protein phosphorylation events control most aspects of cellular function and homeostasis, and the failure of control mechanisms causes disease (Cohen and Tcherpakov, 2010). Most notably, protein phosphorylation events

bacterial infection due to an innate antimicrobial property of MMP-12 in intracellular compartments (Houghton et al., 2009). This MMP was shown to bind to bacterial membranes within the endosomes of macrophages and destabilise bacteria, thus acting as

MMP-9 was one of the first MMPs to be implicated in the mechanism of myocarditis and cardiac dilatation. Heymans et al first showed that inhibition of MMP action through suppression of urokinase-type plasminogen activator, a potent activator of MMPs, and exogenous expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) decreased cardiac inflammation and reduced myocardial necrosis at 7 days, decreasing cardiac fibrosis at 35 days after CVB3 infection (Heymans et al., 2006). When the activity of MMP-2 and -9 were decreased there was a concomitant recovery of cardiac function with decreased immune infiltration. However, shortly after the publication of this paper Cheung et al demonstrated that MMP-9 was in fact a necessary constituent of the antiviral immune response (Cheung et al., 2008). They demonstrated no difference in viral load between MMP-8-/- mice and their WT counterparts. However, MMP-9-/- mice had higher viral loads and virus mediated myocardial damage, during CVB3 infection. Though the antiviral mechanism of MMP-9 action was not explicitly reported, there were significantly elevated levels of interferon-β (IFN-β) in the MMP-9-/- mice. This MMP has been shown previously to cleave and inactivate IFN-β (Nelissen et al., 2003), and IFN-β is known to have a negative feedback effect upon IFN-γ expression (Hanada and Yoshimura, 2002; Yoshimura et al., 2003). The viral loads were similar at early time points (3 – 5 days) post-infection between the genetic MMP-9 knock-outs and WT mice, suggesting that the innate-early immune response was intact, but there were higher viral loads seen in the MMP-9-/- mice 9 days post-infection. These results suggest that there was a deficiency in adaptive immunity in the MMP-9-/- mice, consistent with IFN-β negative feedback inhibition on IFN-γ regulated adaptive immunity in the

**4. Virus induced cell signalling and global aspects of cellular signalling** 

**4.1 Defining cell signalling network models in virus-host interactions** 

of those that are merely coincident to viral infection.

Thus far we have discussed a large number of parallel signalling molecules and pathways that are simultaneously activated during CVB3 infection, from proteolytic death pathways to phospho-signalling kinase cascades that activate a myriad of functions. Systems biology approaches now exist whereby one may survey a large number of pathways and molecules at the same time, query these results using complex statistical methods and elucidate the pathways that are truly required for a particular pathogenic function, and determine which

Host cells have evolved complex systems to detect and eradicate viruses; on the other hand, viruses have evolved mechanisms to compromise essential cellular processes and suppress the host cell defence. In these complex systems, protein phosphorylation events control most aspects of cellular function and homeostasis, and the failure of control mechanisms causes disease (Cohen and Tcherpakov, 2010). Most notably, protein phosphorylation events

**3.5.1 MMPs as modulators of the antiviral immune response** 

an antimicrobial.

MMP-9-/- mice.

**responses** 

mediated by kinases and phosphatases are used by viruses as they are crucial for pathogen replication, propagation, and evasion from host immune responses (Ribet and Cossart, 2010). Several studies have showed that multiple phosphorylated-proteins individually regulate the events that constitute virus replication [reviewed in (Esfandiarei and McManus, 2008)], yet the phospho-proteins are part of an intracellular signal-transduction network, composed of several pathways. Thus, systems perspective studies have enabled us to address network mechanisms active during host-virus interactions, with specific emphasis on elucidating key determinants of disease severity and the effective translation of these concepts into new systems-oriented therapeutic targets.

Virus infection is equivalent to a network perturbation, in that viruses have evolved effective strategies to manipulate multiple signalling pathways and induce crosstalk and feedback loops among pathways to form a network (Garmaroudi et al., 2010). In fact, viruses activate signal-transduction networks through multiple independent events, which include viral docking to receptors, viral protein synthesis, viral progeny release and virusinduced inflammatory responses (Tam, 2006). Thus, to properly understand how signaltransduction networks are disrupted by viruses, a global multivariate approach is required (Ideker et al., 2001; Kleppe et al., 2006).

One of the major challenges of defining a signal-transduction network model in the context of a disease is to study a holistic picture of molecular structures, coming together to make complex and dynamic networks. New tools are required to systematically perturb and monitor signalling processes and functions within cells. Indeed, the emergence of high-throughput, extremely parallel technologies enable biologists to monitor multiple cellular components all together (Albeck et al., 2008). These tools have provided researchers the opportunity to collect comprehensive and large datasets. Nowadays, in the era of "new biology" researchers are 'drowning' in data, in that one emerging challenge is how to organize, interpret and extract pertinent information. The complex web of death pathways discussed in section 2.3 or the complex nature of the immune response discussed in section 3.0, all argue a need for higher level analysis through machine learning. Though this analysis requires skilled computational biologists, using mathematical, statistical and computational techniques to put together the biological components into functional molecular and cellular network models in a systematic fashion (Janes and Yaffe, 2006).

One work that has emerged from recent studies of networked systems in the virusinfected host cell revealed ~260 host cellular factors, affecting virus infection (Brass et al., 2008), however only a small subset of these proteins played a role in the early stage of virus infection. Interestingly, a complementary study showed how a multi-parameter approach could unravel host-virus protein interactions that likely act as a network to facilitate the early steps of HIV-1 infection (Konig et al., 2008). Recently, small-molecule inhibitor pairs were used to perturb pairs of phospho-proteins to reveal causal mechanisms within the signal-transduction network response of cardiomyocytes to coxsackievirus B3 (CVB3) infection. Hierarchical cluster analysis of the resulting dataset showed that paired-inhibitor data was required for accurate predictions of the network. In this study we also depicted a high-confidence network based on partial correlations, which identified phospho-IкBα as a central "hub" in a measured phosphorylation signature (Garmaroudi et al., 2010). Now, biologists are poised to understand the network mechanisms in virus infection, in that these networks might be used to improve patient diagnosis, monitoring, and treatment.

Cellular and Immunological Regulation of Viral Myocarditis 285

consequential. There are also a large number of immune constituents involved in the clearance but also the pathogenesis of myocarditis, from naive T cells to regulatory T cell subsets and a large number of MMPs. Systems biology should prove useful in the near future to elucidate the interactions of proteins and pathways and show the truly pertinent proteins and pathways responsible for cell death, virus replication or MMP mediated tissue remodelling. New questions regarding pathway coincidence and consequence can be investigated. New methods of analysis should assist in the understanding of beneficial vs pathological arms of the immune system. With this knowledge we should be able to design highly useful and robust biomarkers, antivirals and drugs to help direct appropriate repair

Adler, H.S., Kubsch, S., Graulich, E., Ludwig, S., Knop, J., and Steinbrink, K. (2007).

Albeck, J.G., Burke, J.M., Aldridge, B.B., Zhang, M., Lauffenburger, D.A., and Sorger, P.K.

Andrade, F., Roy, S., Nicholson, D., Thornberry, N., Rosen, A., and Casciola-Rosen, L.

substrates: implications for CTL-induced apoptosis. Immunity *8*, 451-460. Aretz, H.T., Billingham, M.E., Edwards, W.D., Factor, S.M., Fallon, J.T., Fenoglio, J.J., Jr.,

Atkinson, E.A., Barry, M., Darmon, A.J., Shostak, I., Turner, P.C., Moyer, R.W., and

Balbin, M., Fueyo, A., Tester, A.M., Pendas, A.M., Pitiot, A.S., Astudillo, A., Overall, C.M.,

Barry, M., Heibein, J.A., Pinkoski, M.J., Lee, S.F., Moyer, R.W., Green, D.R., and Bleackley,

Baughman, K.L. (2006). Diagnosis of myocarditis: death of Dallas criteria. Circulation *113*,

Borisy, A.A., Elliott, P.J., Hurst, N.W., Lee, M.S., Lehar, J., Price, E.R., Serbedzija, G.,

of multicomponent therapeutics. Proc Natl Acad Sci U S A *100*, 7977-7982. Brady, W.J., Ferguson, J.D., Ullman, E.A., and Perron, A.D. (2004). Myocarditis: emergency

skin tumor susceptibility to male mice. Nat Genet *35*, 252-257.

function of regulatory T cells. Blood *109*, 4351-4359.

classification. Am J Cardiovasc Pathol *1*, 3-14.

cells. Mol Cell *30*, 11-25.

*273*, 21261-21266.

Biol *20*, 3781-3794.

593-595.

Activation of MAP kinase p38 is critical for the cell-cycle-controlled suppressor

(2008). Quantitative analysis of pathways controlling extrinsic apoptosis in single

(1998). Granzyme B directly and efficiently cleaves several downstream caspase

Olsen, E.G., and Schoen, F.J. (1987). Myocarditis. A histopathologic definition and

Bleackley, R.C. (1998). Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. J Biol Chem

Shapiro, S.D., and Lopez-Otin, C. (2003). Loss of collagenase-2 confers increased

R.C. (2000). Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol Cell

Zimmermann, G.R., Foley, M.A., Stockwell, B.R.*, et al.* (2003). Systematic discovery

department recognition and management. Emerg Med Clin North Am *22*, 865-885.

and remodelling.

**6. References** 

#### **4.2 Proposing novel therapeutic targets for viral myocarditis through systems biology analysis of host cell signal-transduction networks**

As we have already discussed, cardio-tropic viruses like CVB3 can directly and indirectly further the progression of viral myocarditis to heart failure [(McManus et al., 1993) reviewed in (Marchant et al., 2008)]. To date, there is no curative treatment beyond heart transplantation for viral myocarditis-associated heart failure. Therapies that have been used in patients with myocarditis are immune serum globulin and pleconaril (Pevear et al., 1986; Rotbart, 1999). The anti-picornaviral agent, pleconaril perturbs viral uncoating and in turn blocks viral attachment to host cell receptors. Although, directly targeting viruses has been successful in controlling viral diseases, it suffers from some serious weaknesses, including failure to eliminate chronic viral myocarditis, narrow spectrum of action, and the inherent capacity to force outgrowth of drug resistance mutations (Tan et al., 2007). Thus, the discovery of novel antiviral targets, host cell-based antiviral agents is a more promising approach and deserves more attention (Saladino et al., 2010).

At the cellular level, studies have shown that host cell phospho-proteins essential for viral replication, are potentially targetable [(Marchant 2009) reviewed in (Marchant et al., 2008)]. It is known, however, that phospho-protein networks embrace a system that contains redundant, convergent and even distinct signaling pathways (Borisy et al., 2003). Such combinative properties of signaling networks may counteract the therapeutic efficacy of highly selective drugs due to signalling pathway redundancy. Thus, combination therapy may be necessary to achieve efficacy due to less treatment resistance (Fitzgerald et al., 2006). However, motivation for this initiative, therapeutic synergy of a combination is tempered by concerns about introducing synergistic side effects. Nevertheless, an *in vivo* study that has emerged from recent studies of the healing process in a rat asthma model showed that combining drugs performed better than single ones (Lehar et al., 2009).

Together, to propose novel and promising drugs and therapies requires us to understand network mechanisms at the cellular, tissue and organism level. Using high-throughput technologies in genomics, proteomics, phosphoproteomics, metabolomics and lipidomics, we can develop interactome network models, in that these models ultimately translate to network analysis and data-driven questions in a specific disease context, that can be used to analyse a broad range of different states and disease contexts. There have been recent reports that used systems biology analysis tools in an attempt to untangle the web of kinase signalling pathways that are activated during CVB3 infection of cardiomyocytes (Garmaroudi et al., 2010). There is a current unclaimed niche of research that exists to use similar systems biology tools to analyse the pathways of cell death in order to better understand not only the dominant pathways of virus induced cell death but to also characterise the type of cell death that causes necrotic rupture of infected cells. To perturb specific molecules or biological processes in a combination as directed by network models can be a capable strategy to treat complex diseases.

## **5. Conclusion**

In every facet of CVB3 replication there are a large number of pathways and proteins involved, forming a large network of pathways and complexity too large to analyse by conventional means. The large number of death pathways activated by CVB3 may not all be required to mediate lysis of the infected host cell. The question still remains as to whether there are pathways activated as a matter of coincidence or whether death pathways are all consequential. There are also a large number of immune constituents involved in the clearance but also the pathogenesis of myocarditis, from naive T cells to regulatory T cell subsets and a large number of MMPs. Systems biology should prove useful in the near future to elucidate the interactions of proteins and pathways and show the truly pertinent proteins and pathways responsible for cell death, virus replication or MMP mediated tissue remodelling. New questions regarding pathway coincidence and consequence can be investigated. New methods of analysis should assist in the understanding of beneficial vs pathological arms of the immune system. With this knowledge we should be able to design highly useful and robust biomarkers, antivirals and drugs to help direct appropriate repair and remodelling.
