Enemy at the gate: a novel mechanism of paracrine stress granule induction by viruses

Cells within the body respond to external stimuli in many ways, the most common of which is via the regulation of gene expression. In response to stress such as infection, cells can pause protein synthesis, or translation, and thus the decoding of genetic information, by storing messenger RNAs (mRNAs) away in cellular compartments called stress granules. This defence mechanism allows cells to survive by limiting the use of energy and nutrients that protein synthesis requires until the stress is resolved. It also blocks the spread of viruses as they are dependent on host cell resources to produce viral proteins and replicate. Because stress granules also act as sentinels to sense viral attack, viruses have developed strategies to disable their function.

Stress granules are also of increasing importance because of recent observations that they are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease, as well as resistance to cancer chemotherapy.

Caliciviruses are an important family of small viruses that can cause diseases both in humans and animals. In humans they primarily cause gastroenteritis but also cause general and respiratory infections in animals. Using animal caliciviruses, we previously made significant advances in identifying new mechanisms that viruses use to manipulate the host cell and counteract the cell's defence systems.

Based on our recent results, we are now proposing that in response to virus infection, a novel mechanism allows uninfected cells to activate stress responses and prepare for viral assault. This mechanism is novel and we think it represents a new line of defence against viruses. Therefore, our objectives are to use a combination of cell biology, virology and biochemical methods to 1- characterise the composition of stress granules assembled in response to infection; understand their role in 2- the remodelling of protein synthesis and 3- the antiviral response, and 4- identify which soluble molecules stimulate this mechanism.

From this work we expect to fully understand how viruses regulate the assembly of stress granules, and prime an antiviral state. We can then identify new ways to inhibit virus replication. Therefore, our work will characterise a new mechanism of antiviral response. It will advance our basic knowledge of how stress granules control gene expression and aid in the development of novel antiviral therapies for this important group of viruses, and later other viruses that control stress pathways.

Caliciviruses, a family of small RNA viruses, are important pathogens of humans and animals that have helped us to identify new mechanisms of viral translational control. First, they use a virus-encoded protein attached to the 5' end of the viral genome, namely VPg, to recruit initiation factors and trigger viral mRNA translation. Second, they re-programme host mRNA translation by controlling the activity of eIF4E. Viral infections can also leads to extensive translational control through the aggregation of stalled translation complexes into cytoplasmic stress granules. Stress granules are important for the control of mRNA translation and orchestrating antiviral responses.

We have now obtained present robust preliminary evidence that calicivirus infection results in the paracrine induction of stress granule assembly in uninfected cells. We also demonstrate that these stress granules can be isolated for further functional studies and hypothesise that they play a role in controlling viral infection.

Our aim is to understand how paracrine-induced stress granules contribute to the antiviral response and to characterise a new mechanism of stress granule assembly. First, we will implement a novel affinity purification method to isolate and then characterise the stress granules induced by viruses, using proteomic and biochemical methods. Next, we will dissect the role these stress granules play in the translational control of the host and the antiviral response. Finally, we will identify the messenger molecule(s) responsible for this mechanism.

This work will provide new understanding of the role played by stress granules during infection, and define a new assembly pathway for RNA granules. It may enable new antiviral therapies for the control of viral infection.

The preliminary data presented in this application, and the work planned to build on these findings, will lead to a step-change in our understanding of the role of stress granules in viral infections. This research will have a direct scientific impact in the fields of microbiology and cell biology. It also has strong potential for economic/societal impact. As caliciviruses are important human and animal pathogens, our work may identify new targets for treatment of these economically important infections and therefore has the potential to impact on UK health, society and economy.

Industrial and Economic Impact

The regulation of stress granule assembly/disassembly is a key regulatory process in the response to viral infection. Several studies have suggested that stress granules play an antiviral role by triggering the innate immune responses. Therefore, the stress granule pathway could be harnessed as a strategy to block viral replication. Because stress granules are triggered by the aggregation of host proteins this approach is unlikely to result in the development of drug-resistant viruses. This raises the exciting possibility of developing novel antiviral therapeutics with broad-spectrum activity.

Stress granules are important in several key pathologies. Tumours that produce more stress granules are more likely to metastasise. In addition, chemotherapeutic compounds, such as bortezomib used in the treatment of multiple myeloma can induce the formation of stress granules to inhibit apoptosis in cancer cells. This effect contributes to chemotherapy resistance. Finally, stress granule persistence is linked to several neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and Alzheimer's disease.
A better understanding of stress granule pathways will provide the pharmaceutical industry with new leads in optimising the development of drugs and understanding the mechanisms of drug resistance.

Public sector and Societal Impact

Noroviruses, often referred to as 'winter vomiting disease', are a significant public health problem. Outbreaks in hospitals alone often compromise patient care at a time of year when NHS Trusts are already under pressure, namely the winter months. In addition, norovirus outbreaks in schools, cruise ships, care homes and restaurants have a significant socioeconomic impact with typically 1 million cases in the UK every year. Other caliciviruses are important pathogens of animals with porcine sapovirus causing epidemic outbreaks of gastroenteritis in piglets and feline calicivirus leading to respiratory infections that can sometimes be lethal.
Our research has the potential to deliver impact in the better understanding of these important pathogens of both humans and animals. The findings from our work will be publicised via the University press office and outreach activities to raise awareness in the general public. The PI has already good relationships with the TV and radio channels through multiple appearances locally, nationally and internationally during coverage of the Zika and Ebola outbreaks.

Training of skilled researchers

One postdoctoral research assistant and research assistant will be recruited as part of this project. They will gain extensive expertise in state of the art RNA biology and virology techniques by collaborating with world-leading experts, helping to build their own networks. They will therefore be expertly equipped for challenges relevant to careers both in academic research and industry.