A systems approach to elucidation of protein lipidation during virus infection

Viruses are small infectious agents and are obligate parasites in that they replicate only inside host cells. To cause disease, they must enter specific target cells, overcome barriers to infection and engage with the appropriate cellular machinery which they redirect to make more copies of themselves. This application concerns one of a major group of human viruses, the herpesviruses. These viruses cause diseases ranging from moderate but with high frequency in the population, such as cold sores, chicken pox or glandular fever, through to diseases that while less frequent, can be extremely severe and life threatening, including eye infections, diseases in newborn infants, as well as peripheral and central nervous system diseases. They are particularly dangerous in immunosuppressed patients such as transplant recipients and in AIDS patients. While we have some effective drugs, there are significant limitations with their use and they are effective only against a subset of the herpesviruses. To identify new therapeutic opportunities we must have a thorough understanding of the mechanisms of replication. Such understanding may lead to a rationale way to design novel antiviral agents which may be more broadly effective.
Host cell proteins and viral proteins are modified after (or during) their synthesis with a series of different types of molecules. This process is generally termed post-translational modification and it plays a fundamental role in changing the structure, localisation, stability and function of the potential target proteins. Therefore in terms of virus infection, protein modification can be on host cell proteins and affect their ability to support virus growth or to respond to infection and mount an antiviral attack. Host protein modification can in principle remain unaffected, or specific species can be induced by the host, or by the virus, while other species may be down-regulated. Likewise modification of virus proteins can affect their function, or recruitment into assembly particles, their transport within and out of a cell or their ability to overcome host responses.
One general type of such modification is called lipidation, wherein proteins are covalently coupled to any of a series of lipids (fat like molecules). This represents a key regulatory mechanism in altering proteins and thus regulating processes including membrane targeting, intracellular protein and organelle transport, signalling networks relaying communication from outside to inside the cell, cell structure, metabolic pathways, neurotransmission and immune responses. As such protein lipidation plays a profound role in normal biological functioning and disease progression.
There are four broad types of lipid modification and they can take place on proteins in different combinations, altering the location and function of proteins. However lipidation is broadly less understood than other sorts of protein modification and our knowledge of the range of host and viral proteins which are modified is incomplete. This is because the most frequently utilised approaches lack sensitivity, can be slow to yield results and are not readily amenable to high-throughput analysis exploiting advances in genetic and protein bioinformatics.
We aim to develop novel chemical technologies combined with high-throughput approaches on the role of lipidation of virus and host proteins in herpesvirus infection. Not only will this work yield novel insight into the identity and role of lipidation in this context, it will produce new probes and methodologies available for other systems. It will hugely accelerate our understanding of infection and our ability to perform similar studies in other disease systems not only in infectious diseases. It will enable high-throughput screening of potential new classes of therapeutics and it will provide a roadmap for multidisciplinary working bringing together chemistry and biology to yield novel insight into disease processes.

The first objectives are the chemical synthesis of lipid probes and capture reagents which enable purification and detection of lipidated proteins. Probes for each of classes of lipid modification, will be derivatised with azido or alkyne moieties for subsequent covalent ligation in vitro to the capture reagents. The capture reagents will be derivatised with the reciprocal chemical moieties, and also contain a capture agent (biotin) and a fluorescent detection agent (TAMRA). These moieties enable in vitro ligation of a lipid containing protein to the capture reagent by a copper catalysed chemical reaction. Different combinations of probes and capture reagents will be synthesised, and tested for efficiency of protein incorporation in vitro and in vivo for method optimisation. In vivo incorporation will be assessed by cell extraction, in vitro ligation, capture using affinity enrichment on neutravidin beads, and SDS-PAGE. Lipidated proteins will then be detected by in gel fluorescence of the lipidated protein species. Once initial optimisation is achieved, we will use the best performing probe/capture pairs to examine lipidation on a global scale using nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS). After capture, lipidated species will be subject to tryptic digestion and the peptides analysed by nLC-MS/MS. We will then utilise the optimal reagents and capture pairs for analysis of protein lipidation in vivo during infection and replication of herpes simplex virus, employing stable isotopic labelling in suitable cell culture models coupled with nLC-MS/MS, to produce the first quantitative global analysis of host and viral protein lipidation during herpesvirus infection. Our final objective will be to validate the study and technical platform by individual analysis of candidate proteins by biochemical and molecular approaches using western blotting and introduction of specific cloned species.

The Research Council guidance on how to consider impact and potential beneficiaries states that applicants are not expected to be able to predict impact but to consider who might, potentially, benefit and how. Using the guidance and checklist I submit the following summary for this specific project.

Who might benefit. The public in health and disease.
Why and how. This application is in an important area of infection related to human health and disease. It is at a stage termed early discovery research in a pharmaceutical company setting and while one cannot predict the extent of the impact, there is a real possibility of contribution to human health. However while the most obvious ambition for medical research, this has also the longest time frame and a complex pathway. In limiting impact to the lifetime of the grant, the main contribution is in ensuring success of the research and its dissemination which determine further steps on the longer Pathway, and in being alert to opportunities e.g. in IP or commercial sponsorship as the work develops.

The biotechnology sector.
Why and how. Many companies, across human, animal and plant sectors have interests in development of diagnostic or therapeutic entities. As an example, gene therapy or the delivery of nucleic acids are important applied objectives for a number of companies and indeed herpesviruses are in clinical trials in cancer therapy. Information from this project could be relevant to optimising delivery of viruses or nucleoprotein complexes. While one cannot predict impact in these fields, one needs to be aware of them and the diverse interests in the sector. I am aware of such possibilities and the pathways through combining research, IP, licensing opportunities etc, for creating impact.

Society and skills
Why and how. This research has the direct consequence of training individuals in a broad range of skills, including e.g., the transferable skills in computing and IT, statistics and data handling, problem solving, etc. Such individuals undoubtedly make an Impact in the capabilities they bring to other activities they may embark upon. The Pathway to this Impact requires a secure foundation in which to pursue the research, in-house or external training facilities, mentorship and constructive evaluation. Imperial College has many courses for its researchers, forums for discussion and representation of its scientists and mechanisms e.g., PPDRs to assess progress and aid development for external opportunities. This Impact is achievable over the lifetime of the grant.

Education sector
Why and how. There is a clear need to ensure supply of high quality students in the life sciences. We encourage engagement with schools and pupils, We have dedicated events within for investigators to promote their work. At an individual level, my laboratory accepts work experience students each year. This has stimulated and trained the students and had a real impact on their results and their future careers, usually in a University setting. We routinely help train and supervise undergraduate students in projects or individual tutorials. Catalysed by such practices we also give talks in schools, including primary schools, or help with young pupils and teachers in promoting science.

Science Communications, reporting and the media
Imperial College aims to be an important source of scientific advice, and help create a wide awareness in society of the benefits of research in science and medicine. I am enlisted as a researcher as a point of contact for science and news reporting. Imperial also communicates its research through a variety of routes and encourages investigators to promote their research directly to audiences, for example using social media platforms, and engaging with public audiences. These and other routes have impact in the promotion of research, informed commentators and engagement with internal and external audiences.