Project 13

Impact of virus-induced oscillating stress response on
host cell homeostasis

Ruggieri, Alessia
Dept. of Infectious Diseases – Molecular Virology

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.