Understanding the regulation of stress pathways during viral infections

Background

During infection by viruses, the accumulation of RNA replication intermediates or viral proteins imposes major stress on the host cell. In response to this stress, infected cells can induce several defense mechanisms. In order to promote cell survival and limit the use of energy and nutrients, the stressed host cell induces a global reduction in host protein synthesis. This translational arrest results in the accumulation of mRNAs in cytoplasmic structures called stress granules (SGs). Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. To maintain their gene expression, some viruses have evolved strategies to regulate stress granules response and the assembly of SGs.

Caliviruses contains viruses of both medical and veterinary importance. In humans, norovirus cause acute gastroenteritis with outbreaks in hospitals costing the NHS at least £115 million per year. Unlike human norovirus, feline calicivirus (FCV) and murine norovirus (MNV) can be propagated in cell culture are the models to understand norovirus biology. Using these models, we have obtained preliminary data suggesting that caliciviruses impair the formation of SGs. However the molecular mechanisms responsible remain unclear.

Aims

Using MNV and FCV as model for norovirus, we propose to investigate the molecular basis for SG assembly regulation in infected cells. You will combine the use of cell biology (cell culture, viral infections) and molecular biology (immunoblotting, qPCR and confocal microscopy) to answer the following questions:

a) does SG inhibition occur in eIF2alpha-dependent or independent pathways? Translational arrest can be triggered by various stresses activating different eIF2alpha kinases. Therefore we will investigate whether FCV-infection can disrupt SGs induced via different eIF2 alpha-dependent or independent pathways, using several pharmacological inhibitors of these pathways.

b) Which step(s) during FCV infection mediates inhibition of SG assembly? Our preliminary results suggest that viral attachment/entry is not the step leading to SGs disruption. To unravel the steps in FCV infection that are necessary for inhibition of SGs, we will challenge virus infected for different time points throughout the replication cycle (0, 2, 4, 6 and 8h) and to account for a potential effect of viral RNA replication, cells will be infected in the presence of the viral RNA-dependent RNA-polymerase inhibitor 2-thiouridine. The inhibition of SG assembly in all these conditions will then evaluated by immunofluorescence microscopy.

c) Is a particular viral protein triggering SG disruption? To study the role of individual FCV proteins on SG, you will express individual calicivirus proteins and dissect which regulates SG assembly.

d) Finally you establish the conservation of these mechanisms with other caliciviruses such as murine norovirus and human norovirus.
This project builds on a set of collaboration with the department of pathology at the University of Cambridge and the School of Veterinary Medicine at the University of Utrecht.

Impact and Significance

By understanding how viruses can regulate stress responses and antiviral pathways, this project will identify new drug targets against norovirus, for which no vaccine or antiviral exist.