Functional investigations of the influenza virus proteome.

Viruses are often portrayed as extremely simple infectious agents, but in most cases our efforts to control them have only limited success. This can be clearly seen for influenza viruses, which despite decades of intensive research still cause severe and widespread clinical and veterinary disease, and remain capable of causing devastating pandemics. Viruses are small enough that their replication has to be understood at the level of molecular biology. At this scale, functions are typically carried out through the interactions of proteins. Yet, despite influenza viruses having a limited number of protein-encoding genes (their entire genome could be written out on a couple of sheets of paper) our understanding of how proteins mediate influenza infections is very far from complete. This proposal aims to deepen our understanding of the functions that proteins - those encoded by influenza viruses, and those which viruses co-opt from their hosts - play in an infection.

There are three main reasons why, despite much effort, we do not fully understand the various ways in which proteins mediate viral infections. The first is the remarkable efficiency of viruses in encoding many proteins in a small genome, and in putting those proteins to many different uses. The second is that the function of many proteins can be regulated by chemical modifications ('post-translational modifications') which can act as switches and cause profound changes in their stability, activity or localisation within the cell. The third problem is that we are mistaken when we think of viruses as simple. A viral infection makes use of the thousands of proteins already encoded by an infected cell, with the proteins the virus actually encodes entering this complex environment and steering it towards the production of new virus particles.

I study proteins in viral infections by combining mass spectrometry, a method which can identify and characterise proteins even when in complex mixtures, with molecular biological and virological methods, which can be used to test hypotheses about the function of proteins in infections. During this fellowship I will use this combined approach to search for previously uncharacterised viral proteins and determine new roles for known proteins. I will identify the sites of chemical modification and mutation in viral proteins and determine their effects on function. The information gained through mass spectrometry will allow me to make meaningful molecular biological investigations of complex systems, from the roles of host proteins in forming virus particles to determining the waves of regulatory chemical modifications which sweep through a cell as it enters an infected state.

This work is of fundamental interest, particularly as understanding the intimate association of viruses with their hosts frequently informs the molecular biology of our own cells. Beyond this, the study addresses issues of practical importance. Although I am proposing a programme of basic research, understanding the basic biology of an infection is vital if we are to control it. A detailed understanding of the precise composition of influenza virus particles will be of value to the manufacturers of influenza vaccines, and a better understanding of the regulatory chemical changes in infections will help in the development of antivirals. Antivirals inhibiting such changes have been shown to limit influenza virus replication but their mode of action is unclear, and we need to know which changes the virus relies on in order to improve the action of these drugs.

This research aims to analyse influenza virus infections at a molecular level by combining proteomic studies with molecular biology and classical virology. It has two main objectives: to relate the protein composition of influenza virions to their ability to infect cells, and to relate changes in viral and host protein phosphorylation during the course of an infection to the regulation of viral replication.

The methodology integrates descriptive mass spectrometry (MS) with hypothesis testing in molecular virology. MS will be used (i) for absolute quantitation of proteins in complex mixtures (virions and whole cell lysates), (ii) to map protein phosphorylation and (iii) for relative quantitation of proteins from different time points during infection. I have experience of applying the first two approaches and I have already used them to generate large datasets relevant to the proposal. I also have preliminary data demonstrating the technical feasibility of the third approach. Most importantly, I have experience of combining these large MS datasets with evolutionary, structural and functional data to produce testable hypotheses about virus biology, and I will recruit bioinformatic support to infer hypotheses about more complex cellular pathways. I will then relate protein composition to infectivity by virus titration, protein overexpression and knockdown, studies of interferon induction and (in collaboration) electron cryotomography. For signalling pathways which show changes in phosphorylation during infection, I will knock down or inhibit key proteins in these pathways to test whether they regulate viral growth or the localisation and activity of viral proteins. I will pay particular attention to findings that will inform the development of antiviral kinase inhibitors.

Together, these approaches will provide an opportunity to deepen our scientific understanding of influenza viruses and to develop strategies for their control.

The impacts of the work proposed align with the four components of the MRC's mission.

The proposal tackles a serious threat to human health. Influenza causes thousands of deaths each year in the UK, as well as widespread morbidity. This proposal uses a basic science approach to better understand influenza infections and to identify features through which they may be controlled. In the short term, outputs will be primarily of interest to academia and those engaged in commercial research and development of vaccines and antivirals. In the medium term, the methods developed for this project may inform the regulation of live-attenuated vaccines, as these are currently licensed without a complete characterisation of virion and exosome composition. Characterisation of live-attenuated vaccine composition will also help to assess the risks of vaccination for those with egg allergies. In the long term, improved understanding of the material shed from infected cells may benefit the vaccine industry by identifying more appropriate systems for vaccine growth than eggs, leading to higher-yielding vaccines. This should lead in turn to more cost-efficient purchasing by healthcare providers, and may reduce the time taken to update vaccine formulations, increasing the protection offered to the public. Also in the medium term, the identification of potential targets for antiviral drugs will stimulate research in academia and the pharmaceutical industry. This has the potential to lead to the development and licensing, over the next one to three decades, of novel anti-influenza drugs. Antiviral drugs targeting the host will be less likely to fail through the evolution of resistance. Finally, it is in the nature of basic research that findings without an immediate translational impact can have unforeseeable practical benefits in the future. This is necessarily unpredictable, but it should be noted that the proposal addresses the formation of filamentous virions and the heterocellular suppression of the immune response, two aspects of influenza infection about which we currently know relatively little.

The proposal will support the training of both myself and the PDRA as skilled researchers. The fellowship will support me in developing an independent research career in the UK, and it will provide the PDRA with training to either develop independent research of their own or to transition to work effectively in industry.

The proposal will produce knowledge that is likely to benefit quality of life and the economic competitiveness of the UK. As described above, the research will support efforts to control influenza. As well as offering direct benefits to human health (see above), this may reduce the substantial economic costs of influenza infections to the public and private sectors, which include healthcare costs, absenteeism and losses of livestock and racing animals from veterinary infections. Research related to vaccines and antivirals is also likely to benefit pharmaceutical industries based in the UK, not least because a UK-based company (MedImmune) currently manufactures all live-attenuated influenza vaccines for the northern hemisphere. In addition, work to identify host factors restricting infection may inform, in the medium term, pre-pandemic monitoring of veterinary influenza viruses which pose a risk to humans. A better understanding of the barriers to transmission will help to better focus this vital but costly activity.

Finally, the proposal promotes a dialogue with the public about medical research. As well as the interest and engagement with scientific ideas that may be generated by public dissemination of our findings, I intend for the PDRA and myself to participate actively in science outreach activities (see Communications Plan). As well as engaging with members of the public in the short term I hope that this will foster interest, particularly from potential future scientists, in the medium and long terms.