Systems Biology Approaches for Studying Phosphatase Activity in Coxsackieviral Heart Disease

Viruses are ancient pathogens that infect host cells and hijack their intracellular machinery. Most viruses, like the cardiotropic picornavirus coxsackievirus B3 (CVB3), engage several intracellular signaling pathways during infection through the expression of viral nucleic acid and proteins. During acute infection, these perturbations serve to condition the cell for optimal viral replication and release, while also avoiding immune surveillance. In chronic infections, where virion release is rare, these same perturbations cause long-term cellular dysfunction. For example, when cardiomyocytes become chronically infected with CVB3 they become elongated, resulting in dilation of the left ventricle. This dilation progresses to heart failure and death as the heart’s pumping efficiency diminishes. Studies have shown that the tissue-level consequences of viral infection are not solely dependent on host-pathogen interactions but also on host responses to the environment; specifically, the inflammatory cytokine milieu. During acute infections, pro-inflammatory and antiviral cytokines promote infected cell death and viral clearance. However, prolonged expression of these cytokines is thought to contribute to chronic disease. The work presented in this dissertation asks whether virus-mediated intracellular signaling perturbations influences cellular responses to these inflammatory cues.

We address this question through methods engineering and systems-level experiments. We began by developing a set of quantitative, high-throughput phosphatase activity assays. Protein phosphatases are enzymes that post-translationally modify their substrates to regulate signaling pathway activity. These enzymes can not only affect the activity of multiple pathways but also are important for heart function. Using this assay platform, we show that phosphatase activity is disrupted during the acute phase of CVB3 infection and plays a role in the type I interferon response of infected cells. We then engineer a cellular model of chronic CVB3 infection in cardiac myocytes. Previous models of chronic CVB3 infection utilize non-scalable animal models. By contrast, this in vitro model of chronic infection facilitates subsequent systems-level studies of cellular responses to cytokine stimulation. Using clinically relevant cytokines, we uncover that host transcriptomic adaptions to chronic viral expression result in rewiring of phosphatase responses to inflammatory stimuli. In fact, we observe that stimulus-driven global up-regulation of phosphatase activity diminishes host secretion of proinflammatory cytokines. This work has allowed us to not only understand the consequences of host-pathogen interactions during CVB3 infection, but has also unveiled principles underlying the importance of phosphatases in determining cellular behavior.