Dissecting the mechanism of translational control during calicivirus infection

The caliciviruses are a group of important viruses that infect humans and animals; in humans, the noroviruses are a major cause of gastroenteritis outbreaks, often reported in the UK press as they cause significant problems in hospitals and on cruise ships. In animals, these viruses cause a range of diseases that include a 'flu like illness in cats. The human viruses do not grow well in cell culture in the laboratory so we and others use murine norovirus (MNV) as the best model system to study the human viruses. Production of proteins, or protein synthesis, is an essential process in cells. Messenger RNA, or mRNA, contains the information that is decoded into proteins by the host cell protein synthesis machinery called ribosomes and is assisted by a number of protein factors termed initiation factors. One of the key factors involved in this process is named eIF4F and consists of three proteins, eIF4E (that binds to a structure on the mRNAs called a cap to recruit the ribosome), eIF4G (a scaffold protein that bridges the eIF4F complex to the ribosome) and eIF4A (which helps unwind any structure in the mRNA to allow the ribosome to move along it). In order to make new virus particles, viruses must manufacture new virus proteins but they rely on using the host cell's protein synthesis machinery to do this. We have previously demonstrated that caliciviruses use a novel mechanism for synthesising new virus proteins in infected cells. We have made a number of significant advances in the understanding of how the caliciviruses produce viral proteins: i) We have shown that a viral protein called VPg that is attached to the end of the viral mRNA instead of a cap, binds to one of the key proteins in the cell required for protein synthesis, eIF4E. These viruses have therefore evolved a novel 'proteinaceous' cap substitute that mimics the 5' end of a cellular mRNA. ii) We have also shown that other host translation initiation factors (eIF4A) are required for calicivirus protein synthesis and interfering with these proteins inhibits virus replication. iii) Although feline calicivirus and murine norovirus share the common mechanism of VPg-directed protein synthesis, some fundamental differences seem to exist in their requirements for the cellular initiation factor proteins. iv) Our recent work has shown that these viruses can manipulate the eIF4F complex to regulate production of viral proteins. Building on these findings and our expertise, we now wish to carry out a comparative analysis of the process of calicivirus protein synthesis using modern biochemical techniques, with the aim of understanding the cellular factors they require and how they modify these proteins to aid the production of virus proteins. Specifically we will: 1) Use advanced biochemical techniques to identify what cellular initiation factors are found in complex with the calicivirus VPg protein and then investigate what role they play in virus protein production. 2) Analyse the effect of calicivirus infection on the host eIF4F complex to fully understand how the virus can modulate it to its own advantage. This will tell us how these viruses manipulate the host cell to ensure efficient production of viral proteins. If we can fully understand the mechanism of calicivirus protein synthesis, we can identify ways to inhibit virus replication, and so this work will ultimately aid in the development of novel antiviral therapies for this important group of viruses.

Caliciviruses are important pathogens of man and animals, such as the noroviruses which cause major outbreaks of gastroenteritis. We have previously demonstrated that translation initiation on calicivirus mRNA is dependent on the viral protein (VPg) linked to the 5' end of the viral mRNA and that VPg acts as a novel proteinaceous 'cap substitute' to attract ribosomes to the viral mRNA, via its interaction with the cap-binding protein, eIF4E. We have also started to analyse the requirement for other components of the eIF4F complex for FCV and MNV mRNA translation; while we have shown that eIF4A is required for both FCV and MNV translation, the viruses may differ in their requirements for eIF4G. Therefore, our first aim is to conduct a comparative analysis to identify and functionally characterise initiation factors within the FCV and MNV VPg-initiation complexes. We will first use a proteomics approach to identify initiation factors contained within the VPg-initiation complexes and then use a number of complementary biochemical techniques to assess their functional role in VPg-directed translation initiation. Furthermore, we have recently demonstrated that both viruses modulate the eIF4F complex during infection, particularly the activity of eIF4E, and propose that these changes are involved in regulation of viral translation. Therefore, our second aim is to analyse biochemically the effect of calicivirus infection on the eIF4F complex, by characterising the signalling pathways involved in phosphorylation of eIF4E and its regulating protein, 4E-binding protein-1, by using quantitative proteomics (SILAC) to measure the changes to eIF4F during infection and finally to assess the effect of these changes in regulating calicivirus protein synthesis. Understanding the mechanism of calicivirus protein synthesis may lead to the identification of novel antiviral targets for this important group of viruses.

We are uniquely placed to allow us to fully characterise the initiation factor requirements for translation initiation on calicivirus RNAs. This family of poorly characterised viruses are not only responsible for important human (e.g. norovirus gastroenteritis outbreaks) and animal (e.g. feline calicivirus infections) diseases, but were recently highlighted as a major biopharmaceutical contamination problem. Vesivirus 2117 (related to feline calicivirus) contamination of the bioreactors of Genzyme, resulted in a gross loss in excess of $300 million in 2009. In the short term, the primary beneficiaries of our work will be those academics working in the translational control, cell signalling and virology fields, as we identify novel pathways of translational control and regulation of virus replication. Primarily this will be achieved through publication in high impact, open access journals and through presentation at national and international meetings. However, in the long term, a wider group of beneficiaries from the commercial sector, such as the medical pharma industry, veterinary medicines industry and perhaps even the travel industry (given the highly publicised norovirus outbreaks on cruise ships), may ultimately benefit from our work. As we dissect an important step in virus replication, production of viral proteins, we may identify a novel mechanism by which we can inhibit calicivirus replication and hence, our results may be of significance to those searching for novel antiviral strategies against these important medical and veterinary pathogens. We expect our work from this project to have an impact towards the end of the three year project and beyond, should we identify any potential targets for rational drug design. It is at this stage that we would aim to engage potential commercial audiences. Given that most of the pioneering research on understanding calicivirus translational control has arisen from our labs, we expect that our continued collaboration will help underpin the UK's competitiveness in this area.