What are the molecular mechanisms underlying the roles of the genome-linked virus protein (VPg) in calicivirus replication?

If you have ever been on the receiving end of a 'winter vomiting bug' infection, you will be well aware of the debilitating effects of the human norovirus, one of the main types of calicivirus. The combination of vomiting, diarrhoea and fever is extremely unpleasant. Though rarely fatal, noroviruses inflict a significant economic burden in terms of work absenteeism and the costs of hospitalisation (where the virus is often spread). Other caliciviruses infect domesticated animals and can harm the productivity of the biotechnology industry. Though simple in construction - caliciviruses consist of a protein shell surrounding a single molecule of RNA that encodes the virus genes - these pathogens have a complex replication cycle. We are particularly interested one of the smallest viral proteins, VPg, which is chemically attached to the start of the virus's RNA genome and has two unusual, but critical, roles in the virus replication cycle. Using a mechanism that has only recently been discovered and is still not well understood, VPg directs the viral RNA to the cell's protein synthesis machinery, as part of a covert operation to trick the cell into making virus proteins. Once new virus proteins have begun to accumulate, VPg can assume its second critical role, which is to initiate the process of making copies of the viral RNA for packaging into new virus particles. It does this by binding to the virus polymerase and serving as the attachment point for the synthesis of short chains of RNA that form the primers needed to initiate copying of the RNA genome. We propose to investigate how exactly VPg contributes to the initiation of viral protein synthesis and RNA replication by focusing on the mouse norovirus (MNV). This is an important model system for human norovirus infection, since it has a similar mode of replication and causes a very similar pattern of infection. The study of MNV is all the more important since it has so far proved impossible to work with human noroviruses in the laboratory. We have recently made significant breakthroughs by determining the structures of VPg from MNV (and from a related virus, feline calicivirus) and of the MNV polymerase. The VPg structures were a particular surprise since they had been widely supposed to be unstructured. However, they are extremely informative and have immediately suggested a novel hypothesis of how VPg interacts with the polymerase to kick-off RNA copying: we propose that VPg must unfold to interact productively with the enzyme. Moreover, one of our collaborators (I.G. Goodfellow) has recently shown that MNV binds to the middle domain of eIF4G, a key component of the cellular protein synthesis machinery. This interaction directs the translation of the viral RNA (to which VPg is attached) into the proteins needed for viral replication. These exciting preliminary results put us in a uniquely advantageous position to launch a new program of investigation that will significantly advance our understanding of how VPg works at the molecular level in driving calicivirus replication. We will round out our structural analysis of VPg by determining the structures of human norovirus VPg and that from RHDV (another type of calicivirus) that is deadly in rabbits. Our preliminary structural results have allowed us to design a program of work to fully characterise the mode of action of VPg in RNA replication (Does it unfold? What parts of VPg bind to the polymerase? Does the viral RNA aid that binding?). These experiments should enable us to determine the structure of the VPg-polymerase complex, the ultimate test of our mechanistic hypothesis. Finally, we will also be able to analyse in detail the interaction of MNV VPg with the middle domain eIF4G, though binding experiments and structural analysis by NMR or protein crystallography. In total our plan of investigation will provide a new level of understanding of key aspects of calicivirus replication.

Caliciviruses are important pathogens of humans and other animals. Noroviruses, the calicivirus genus responsible for most infections in humans, are a major cause of non-bacterial gastroenteritis and cost the UK NHS in excess of £100m annually. Work to understand human norovirus infections has been hampered since is has not yet been to replicate these viruses in tissue culture or in tractable animal models. Fortunately, useful animal models have become available recently, most notably murine norovirus (MNV), which have allowed investigation of calicivirus infection at molecular, cellular and whole animal levels. Our proposed program of research aims to dissect the molecular roles of VPg, a virus protein that is covalently attached to the 5'-end of the RNA genome and has critical, but poorly characterised, roles in the initiation of both translation and RNA replication. In preliminary work we have determined the structures of VPg from MNV and feline calicivirus, revealing unexpected helical cores that differ very surprisingly between the two proteins) and the structure of the MNV polymerase. Moreover, our key collaborator (Goodfellow, Imperial) has discovered a specific interaction between MNV VPg and the middle domain of eIF4G, which appears to be critical to virus translation initiation. These findings mark a turning point in the investigation of the critical roles of VPg in calicivirus replication. They have led to the formulation of new hypotheses of VPg structure and function that we are uniquely well placed to investigate. Our program will significantly extend the analysis of calicivirus VPg structures and will use our new structural information to reveal - through carefully designed functional and structural analysis of the relevant VPg-protein complexes - how VPg interacts productively with the viral polymerase and with eIF4G to aid propagation of the virus.

Specific to users/beneficiaries outside the academic research community. This proposal is primarily a piece of fundamental research and will have a profound influence on the investigation of calicivirus replication. Although the calicivirus family includes human noroviruses and the proposed work will focus on murine noroviruses, the best available model system for human infections, the impact or our research beyond the community of scientists is more difficult to predict. Human norovirus infections are a worldwide problem and cause a significant burden of disease in the UK. Our program of research will provide new insights into two key steps in the pathway of calicivirus replication. In particular, it will dissect the interactions that the calicivirus VPg makes with the viral polymerase during RNA replication and with eIF4G during the initiation of virus protein synthesis. It is conceivable that the interactions that VPg makes with these proteins may be revealed as viable targets of antiviral therapy (for example, by the development of drugs that would prevent binding of VPg). Although pursuit of this line of investigation is not an immediate goal, should such a possibility arise, we will explore its investment potential with IC Innovations. Our work will also be of interest to the general public since human norovirus infections are well established in the public mind (and the public gut) as 'the winter vomiting bug'. This notoriety will provide a valuable opportunity to lever reports of our work into popular media outlets (see below). Communication & engagement The primary channel for dissemination of the results of our research program will be through the peer-reviewed scientific literature. However, to reach the broader public we will use more accessible channels. I have been an enthusiastic proponent of public engagement on scientific issues. For the past two years I have written a regular science blog, called Reciprocal Space. This was initially hosted at Nature Network but has recently moved to Occam's Typewriter (http://occamstypewriter.org/scurry) in an effort to expand the readership beyond the scientific community. My blog provides a frank and accessible window into the working life of a modern-day scientist. I has also produced a video about his virus research on FMDV and spoken about it on the Diamond Podcast and to schoolchildren, both in schools and as a winning participant in the 'I'm a Scientist, Get Me Out of Here' competition (funded by the Wellcome Trust). He has contributed to discussions on science both online and in the national press and was one of the organisers of the Science is Vital campaign that help to stave off cuts to the UK science budget in Oct 2010. (See Impact Plan for a full set of links). These activities will be continued through the period of this project and will absorb, where possible, aspects of the work to be done on calicivirus replication (e.g. spot reports on progress, video of summary of a key result). The PDRA employed on this grant will be encouraged to participate fully in these activities. Exploitation Though not an immediate goal of our present program, as mentioned above, we will be mindful of the potential of our work to open up the possibility of developing VPg-protein interactions as a target for drug development. This is not a trivial undertaking since, in addition to the planned determination of high-resolution structures of VPg-polymerase and VPG-eIF4G complexes, it would require development of an assay of VPg activity that can be formatted for high-throughput. Should our results lead us in this direction, we will seek additional funding to carry this work forward, possibly by building on existing links with the Drug Discovery Unit at Dundee University with whom Prof Curry is has collaborated on a drug screening program targeted at the 3C protease from foot-and-mouth disease virus (funded by DEFRA).