Summary Project description 2nd funding period
The overall goal of this project is to study how hepatitis C virus (HCV), a major causative agent of chronic liver disease, establishes fine-tuned interactions with host stress responses to allow productive infection. Cells respond to several forms of stress, including viral infections, by repressing global protein synthesis. Messenger RNAs (mRNAs) encoding non-essential proteins are silenced into stress granules (SGs), cytosolic aggregates of stalled mRNAs, to allow the selective translation of mRNAs required for the restoration of cellular homeostasis. We previously discovered that HCV triggers a highly dynamic oscillating stress response that can be visualized by cycles of assembly and disassembly of SGs. SG oscillations correlated with prolonged survival of infected cells despite extended stress duration. In the first funding period, we characterized in detail SG main regulators by combining single-cell and bulk population approaches. Based on these results, we generated a stochastic mathematical model that identified the molecular determinants involved in HCVinduced SG oscillations. We could experimentally validate that different SG oscillation patterns arise through variations of viral trigger and stress kinase concentrations.
In the next funding period, we will build on these findings to assess how SG oscillation is coordinated with cell homeostasis. First, we aim at determining the function of SG oscillations and understand whether they might influence the survival of HCV-infected cells. To this end we will exploit the mathematical model to predict how different SG oscillation frequencies impact on the cell fate decision process. These predictions will identify key regulators that will be experimentally validated using the previously developed three-color long-term live-cell imaging. Second, we want to study the dynamics of the stress response to HCV infection in physiologically relevant systems. In collaboration with Cavalcanti-Adam and Dao Thi, we will establish cell culture systems with increasing complexity such as 3D polarized hepatoma cells and human stem cell-derived hepatocyte cultures. We will adapt these cultures for live-cell microscopy in collaboration with Fackler. SG assembly and disassembly are multistep processes that remain poorly understood. So far, HCV infection is the unique cellular stress response in which these processes occur repeatedly resulting in SGs whose size, shape and number vary over time. Taking advantage of the large biophysics expertise offered in the consortium, in the last aim, we plan with Schwarz and Tanaka to determine the mechanical forces and energy consumption necessary for SG assembly and disassembly over time. In collaboration with Spatz, we will try to apply a bottom up approach and use droplet stabilized Giant Unilamellar Vesicles to study the minimal requirements and dynamics of the SG assembly process.