Methods Development for Quantifying Dynamics of Host-Viral Interactions

Systems biology approaches biological complexity with the underlying assumption that a living organism is more than the sum of its parts. At the cellular and molecular scale, treating signaling events as an interconnected network, rather than isolated events, is fundamental to systems biology. Protein phosphorylation is a major posttranslational modification that alters the activity of enzymes. The regulation of protein phosphorylation is critical for signal transduction. The addition of phosphate to a protein is catalyzed by protein kinases. Kinase activity is itself regulated by posttranslational modification. Given this complex regulation, methods to directly measure protein kinase activity are desirable. To understand these complex networks, researchers need tools to gather measurements across the network. Current direct measurement methods have the drawbacks of being low throughput and requiring large amounts of cellular material. To address these issues, we have developed an ultrasensitive multiplexed activity assay for cellular kinases which increases throughput five-fold while reducing the cellular material required by over an order of magnitude. This improvement will allow for studies to more broadly look at cellular networks.

Cellular network dynamics are impacted by many, if not all, diseases. For example, viral infection creates a dynamic network within the host cell, connecting the viral life cycle to the host-cell’s processes. Specifically, the cardiotropic picornavirus, coxsackievirus B3 (CVB3), impinges on the host-cell’s networks as it establishes an infection and attempts to evade immune detection. The dynamic network of interactions between the virus and host-cell result in complex feedback loops whose study would be aided by the development of a mechanistic computational model. To date, no computational model of CVB3, or a closely related virus, exists that captures the entire life cycle of the virus from entry to encapsidation. Here we seek to model the entire life cycle of CVB3 within a cardiomyocyte as well as the interaction between the host-cell’s innate immune response and the viral antagonism of that response. The understanding of these complex interactions will allow for the discovery of novel biological insights.

Insights into the interactions between CVB3 and the host-cell have been studied in the past, however few have taken a systems biology approach. Our lab utilized a high-throughput assay to measure the activity of phosphatases, which catalyze the removal of phosphates from proteins, to characterize the impact of CVB3 infection on the host-cell phosphatases in the context of interferon stimulation. The results of this study pointed to an interaction between nuclear c-Jun N-terminal Kinase (JNK) activity and CVB3 replication. Using pharmacologic and genetic approaches, we demonstrated that increasing JNK activity resulted in an increase in CVB3 protein synthesis. This work demonstrates the value of novel assay development and systems biology approaches to understanding complex interactions such as those between CVB3 and host-cell networks.