Understanding the reprogramming of host mRNA translation during calicivirus infection

Our overarching aim is to understand the mechanism by which a group of poorly characterised, yet important viruses, regulate host gene expression to modulate how cells respond to viral infection

Cells within the body respond to environmental stimuli and pathogen infection in many ways, the most common of which is via the regulation of cellular gene expression. The expression of the information stored in our genes is tightly controlled at numerous levels, to ensure that the correct protein is produced at the right time and quantity. First, the genetic information encoded in our DNA is converted into messenger RNA (mRNA), via a process called transcription. The mRNA is then "translated" into proteins by structures referred to as ribosomes, assisted by proteins called initiation factors. eIF4E is one of these proteins and its key role is to direct the ribosomes and other proteins to the mRNA. This process is called translation and the synthesised proteins make our cells what they are, defining their properties and functions. Importantly, changes to the proteins in our cells also help to fight against invading pathogens such as viruses.

While viruses can infect most organisms and cause severe damage, they consist primarily of RNA or DNA enclosed in a protein coat and lack the factors required for replication and dissemination. They are therefore dependent on the host cell resources to produce viral proteins. Thus, many viruses have developed strategies that regulate the function of the host protein synthesis machinery, often leading to preferentially translation of viral mRNAs.
Caliciviruses are a family of small viruses that can cause diseases both in humans and animals. In humans they primarily cause gastroenteritis. While the human norovirus, a calicivirus, does not grow well in cell culture, murine norovirus, a mouse homologue, acts as model with which to study many aspects of calicivirus biology in the laboratory. It represents an excellent model to dissect how viruses affect the translation of host cell proteins. Using this model we have previously made a number of significant advances in the understanding of how the caliciviruses produce viral proteins. We found that a virus-encoded protein, called VPg, is essential for the translation of viral mRNA as it coordinates the recruitment of host proteins to the viral RNA. We also showed that calicivirus infection modulates the composition and activity of the host translation machinery. However, we still know very little about how pathogens in general, and caliciviruses in particular, modulate the translation of host mRNAs during infection. This is important because understanding the modulation of specific host mRNA translation by viruses can reveal how viruses manipulate the organism's response to infection.

Our hypothesis is that caliciviruses alter the translation of specific mRNAs in the infected host to regulate antiviral gene expression, and that they do so by modulating the translation factor eIF4E. Therefore, our objectives are to use high-throughput sequencing and biochemical methods to 1- characterize how the viral infection alters the profile of mRNAs that are translated by the infected host and 2- to understand how the activity of eIF4E is regulated by caliciviruses during this process.
If we can fully understand how caliciviruses control the activity of translation factors and reprogramme the host protein synthesis, we can identify ways to inhibit virus replication. Therefore, our work will aid in the development of novel antiviral therapies for this important group of viruses, and perhaps other viruses that regulate translation. Understanding the fundamental mechanisms of gene regulation is important not only for virologists but also for broader academic communities. By advancing our basic knowledge of translational control we may understand better several pathologies that are linked to modulation of eIF4E activity, such as cancer and diabetes.

Caliciviruses, a family of small RNA viruses, are important pathogens of man and animals yet they remain poorly characterised. They use a novel mechanism of viral protein synthesis that involves the recruitment of cellular initiation factors to a virus-encoded protein attached to the 5' end of the viral genome, namely VPg. We have recently discovered that calicivirus infection regulates the activity of eIF4E via phosphorylation. We also found that host mRNA translation undergoes a specific reprogramming as a result of p-eIF4E associating with polysomes. Our preliminary data would indicate that this biased recruitment is focused on mRNAs involved in the cellular response to viral infection and may facilitate virus replication.

Our overall aim is to dissect how norovirus infection alters the gene expression profile of infected cells contributing to viral pathogenesis, and how the regulation of eIF4E activity participates in this response to infection.
First, given our observations on the translation of specific genes, we will characterize the global regulation of the host mRNA translation by using polysome profiling and RNA sequencing. We will confirm our analysis by using proteomics to further characterize the impact of this translational control on the antiviral response during calicivirus infection. Then, we will unravel the specific contribution of eIF4E phosphorylation in this process. We will define the function of eIF4E phosphorylation by identifying mRNA specifically translated by p-eIF4E using polysomes profiling and animal models. Finally, we will establish the contribution of the kinase responsible for eIF4E phosphorylation, MNK, and characterize the interactions between MNK-eIF4G-eIF4E and VPg.
This will provide an unprecedented understanding of the regulation of host gene expression by caliciviruses, from mRNA production to protein synthesis, and shed light on how these viruses may modulate the antiviral response.

The preliminary data presented in this application, and the experiments planned to build on our findings, will lead to a step-change in our understanding of the control of host translational and the host antiviral response in calicivirus infection. Our findings are also likely to shed light on fundamental processes underpinning the control of gene expression and the host response to infection. This research will have a direct scientific impact in the fields of virology, translational control and host/virus interactions. 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 translation initiation and the signalling pathways associated with translation are well established targets for therapeutic intervention against diseases that arise as a result of translational deregulation. The MAPK pathway has already been the focus for the development of drugs against cancer (e.g: Selumetinib for small cell lung cancer or Trametinib for metastatic melanoma). In addition, numerous cancers have been identified in which eIF4E is over-expressed and/or phosphorylated. The dissection of the points in which translation is regulated during viral infection will therefore provide the pharmaceutical industry with new leads in i) the development of specific antiviral therapies and ii) the development of drugs for pathologies linked to deregulation of translation, such as cancer. Importantly for the pharmaceutical industry (Pfizer, GSK, AstraZeneca), some of the drugs that are currently being developed to control cell proliferation (e.g: Selumetinib, Trametinib, PD0325901, LY2228820) could also act as potent antivirals through the control they exert on eIF4E phosphorylation. This could expand the portfolio of application for existing drugs, greatly reducing the development time, benefiting the UK and worldwide population.

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. The 2012-2013 winter season saw in excess of 1 million cases in the UK, which is typical of the annual norovirus season. The findings from our work will be publicised widely via the respective university press offices but also via our outreach activities raising awareness of noroviruses in the general public.

Training of skilled researchers
Two research assistants, one at post-doctoral level and one graduate RA, will be recruited as part of this project. Both will receive extensive training in state of the art molecular methods for the study of gene expression (RNA sequencing, polysome profiling for PDRA1 and proteomics for RA2). They will also be trained extensively in molecular biology and virology techniques. They will gain experience in the systems biology-type approaches that integrate large data sets. We aim to provide a holistic set of skills that will equip the research assistants for challenges relevant to a wide range of careers both in academic and industrial research, increasing their long-term career prospects. In addition, our laboratories regularly host both undergraduate and post-graduate students, who will also benefit from exposure to the BBSRC-funded research.