A structural and functional characterisation of the L-polymerase replication complex of the Monanegaviruses

To understand how biological processes take place, and to be able to control them, knowledge of the 3-dimensional shape of the molecules involved is very important. Indeed, it is the shape of a molecule that determines what it can do and how it interacts with other molecules.

Viruses, essentially, are small capsules containing genetic material. This genetic material encodes the information required to make a copy of the virus. Viruses infect cells (of humans/animals/plants/bacteria) and multiply inside these cells, mostly killing them in the process, and causing damage to the organism. To multiply, viruses need to make copies of their genetic material and also produce viral 'proteins' (fairly large molecules with a specific shape and size, for which the blueprint is provided by the viral genes). Some of these viral proteins are more or less passive entities that combine to form the viral capsules. Other proteins function as 'enzymes', meaning that they have the ability to actively 'do' things, such as stringing together new copies of the virus genetic material from essential building blocks.

We are studying a large group of viruses (called Mononegavirales) which has several members that are highly infectious and/or dangerous to humans (for instance Ebola virus, Rabies virus and Measles virus). These viruses produce a large enzyme, called the L-polymerase (L simply signifes that it is a Large protein), that is instrumental in the replication of their genetic material and in 'reading' the viral genes (the blueprints), the first stage in protein production. As viruses cannot replicate without properly functioning L-polymerases, understanding how these work can allow us to find ways of blocking them, and stop propagation of the virus. Indeed many of the anti-viral drugs against viruses such as Herpes and HIV, currently on the market specifically target the viral polymerase and prevent it from functioning.

Here we propose to find out what the 3-dimensional shape is of this L-polymerase from these human pathogenic viruses. For this, we will produce the L-polymerase protein (and specific portions of the protein) in large amounts, using cell culture systems, and this material will be crystallised. X-rays can then be used to visualise the protein that forms the crystals and to determine their shape in atomic detail. This will lead to a better understanding of how the L-polymerase protein works, and will help us identify ways of blocking its activity. For example, it would enable the design of small molecules with a shape complementary to that of the important part of the L-polymerase where the genetic material is replicated. These small molecules would then bind to the L-polymerase stopping it from working thereby blocking virus replication.

The Mononegavirales order of negative-sense single-stranded RNA viruses comprises many significant human pathogens. The L-polymerase (MW ~250kDa) is key to the replication of these viruses, and is an ideal drug target. No structures of L-pol or domains from Mononegaviruses have been published and molecular mechanisms are not understood. We recently solved a C-terminal domain of L-pol of hMPV (CR-VI+), and showed that it is responsible for cap synthesis. We propose to capitalise on this success, by structural and activity studies on L-pols of other, medically important mononegaviruses, whilst also carrying out more in-depth analysis of CR-VI+ of hMPV (and other members of the pneumovirinae subfamily).

Bioinformatics will inform the decision on which representatives from a large pool of L-pol genes are likely to express. Synthetic genes will be used, optimised for our eukaryotic expression systems (often crucial in the expression of "difficult to express" proteins). Small-scale expression tests will fine-tune the construct boundaries, and select the most suitable expression systems, prior to/in preparation of large-scale protein expression, purification and crystallisation.

Crystals will be analysed at Diamond Light Source (Harwell, UK). Once suitably diffracting crystals are obtained, standard methods will be employed to solve the structures. Recombinant protein will also be used for relevant activity studies (in vitro polymerase-, phosphohydrolase-, guanylyltransferase-, methyltransferase- and polyadenylation assays), in collaboration with Dr E. Decroly at the AFMB ( http://www.afmb.univ-mrs.fr). Testing of structure based hypothesises and the effect on virus replication, using cell-based reverse genetic systems, will be done in collaboration with Dr B. van den Hoogen (Erasmus MC) and Dr H. Bourhy (Pasteur Institute). Cryo-EM will be utilized for imaging of L-pol, and crystal structures will be fitted into 3D EM reconstructions

Members of the Mononegavirales order are major human pathogens that seriously affect human well-being (morbidity and mortality) and represent a major economic burden on healthcare. For some of these viruses, the case fatality rates are alarmingly high. For example, the WHO reported 2299 cases of Ebola from 1976 to 2011, 67% of which were fatal; deaths caused by rabies are estimated at >55,000 per years. Even for established viruses, such as Measles, the WHO estimates >150,000 deaths per year. These viruses tend to emerge and remerge from animal reservoirs, and some viruses, such as Nipah Virus and Hendra virus, can cause serious animal epidemics, with devastating economic consequences for the farming communities and countries involved. There is currently very little known about the P-protein mediated interaction between the L-polymerase and the viral N-RNA complex, and how the molecule coordinates and choreographs its functions as it replicates its genome and transcribes viral mRNAs. The research proposed would address these fundamental questions that are key to understanding viral replication.
Although the proposed research is strongly rooted in basic science, the findings are expected to make an impact on human health in the long term. These viruses affect large human populations across both the world's poorest and richest regions. Basic research, as outlined in this proposal, will contribute key knowledge required for drug design and vaccine development. For example, the solved structure of the L-polymerase (or functional portions) of Rabies or Ebola virus will provide valuable information for the development of specific anti-viral therapies by rational drug design. Thus mid-term beneficiaries will be in research and development teams in the pharmaceutical industry and in governmental organizations. If their drug and vaccine developments will be successful, in the long term the beneficiaries will be infected patients in the endemic areas. Furthermore, vaccination against Rabies or Ebola would enhance the quality of life, health and economy in these areas and provide protection to hospital personnel.

Dissemination of outputs: A clear priority for the applicants and the project as a whole will be active engagement with various stakeholders. For 'scientific impact', the academic community will benefit, through meetings, conferences and published scientific outputs (e.g. scientific papers and structural datasets), providing a detailed understanding of the structural mechanisms that regulate genome replication and transcription. These results will also help in understanding the evolutionary relationship between members of the seemingly diverse Mononegavirales order.
The Division of Structural Biology is part of a wider interconnected multidisciplinary EU FP7 consortium (SILVER) working together on the structure, molecular biology, surveillance and control of disease caused by emerging RNA viruses. The study, by way of targeted meetings and publications, will engage other scientists and other stake holders. For societal and political impact, the work will help inform vaccine and diagnostic-assay development, and possibly antiviral drug design. The applicant and collaborators will provide advice to both UK and European government agencies (via DEFRA and the EC) concerning the surveillance, monitoring and control of particular mononegaviruses that would threaten human and animal health in the UK and in Europe.

Impact on skills and training: The postdoctoral scientist and any externally funded post-graduate students will be given training in a range of biological and physical techniques that will benefit their future prospects in academia or industry, particularly in molecular biology, proteomics, biophysics, electron microscopy and crystallography skills.