Structural Studies on Viruses, Viral Proteins and Cell Interactions

Viruses vastly outnumber cellular organisms, but reproduce inside cells, and are often very difficult to combat. Although virus diseases such as 'flu and the common cold have been with us for many years, there is always the risk of new viruses emerging as major threats to human health. The 2014 Ebola epidemic is a powerful example of such an emergent virus infection and the belief underpinning my research proposal is that a proper structural/functional understanding at the molecular and atomic level of the main lineages of viruses will provide fundamental knowledge to inform our therapeutic responses to both emerging infections and also to well known (re-emerging) virus diseases such as Hand-foot-and-mouth disease, a disease of children which is an especial problem in East Asia. Whilst disease control programmes will provide front-line defence, the fact that Smallpox and Rinderpest have been eradicated shows the power of well organised global vaccination programmes, combined with effective vaccines. Vaccines work by the recognition at the molecular level of the virus capsid and I believe that vaccine development is ready for a major revolution. The highly effective methods devised fifty years ago, based on chemical inactivation or extensive passage to deliver attenuated virus strains, might now be supplanted by the delivery of viral-like particles (VLPs) made using recombinant DNA technology so that can be highly immunogenic and yet safe. Many aspects of the requirements for such particles, for instance, correct assembly, appropriate thermal and chemical stability, can, in principle, be engineered into such particles, guided by knowledge of the atomic level structure. The other therapeutic approach to viral diseases has traditionally involved either small molecule drugs or biological agents, especially antibodies, to either prevent or treat an infection. Due to the high cost of clinical trials the delivery of such therapeutics is ultimately done by large industrial concerns and the role of academic researchers is usually limited to the pre-competitive stage. Indeed the work I propose here is primarily to develop our underpinning knowledge of the structure and function of viruses, and will mostly not in the short term lead to direct benefits to human health. However in the longer term such basic work finds applications, sometimes by opening up un-thought of therapeutic opportunities.

In this broad context my programme aims to use the latest methods of structural analysis, especially electron and light microscopy and X-ray diffraction, to piece together a better understanding of how several viruses work. The major group of viruses are the picornaviruses, which include a range of human and animal pathogens, from agents of the common cold, through polio to hand-foot-and-mouth disease to hepatitis A. I will also try to illuminate the structure of the small particles of the hepatitis B vaccine and to understand how viruses such as human rotavirus (a major cause of infant death in poorer countries) function as rather complicated replicating machines. To enable this we will also develop some cutting edge methods to deliver improved analyses. Most of the viruses that I propose to work on are from virus families that include important human pathogens, however I will also explore some non-mammalian viruses, since I believe there is still untapped potential, for instance, to intervene with bacterial viruses in the fight against drug resistant bacteria.

My research plan for the next five years aims to extend, from a structural perspective, our basic understanding of certain viruses, many of which are of direct relevance to disease.

In addition we will, as appropriate, explore how aspects of such fundamental understanding can be used to open up avenues to novel anti-viral therapies and vaccines.

Given the ongoing revolution in integrated structural biology, from super-resolution fluorescent microscopy, through electron imaging to X-ray scattering and diffraction we aim to answer fundamental questions in virology. To do this we plan, as required, to develop and extend methods. This more technical work may provide methods of utility to other researchers.

The virology questions addressed fall into four areas, with methodological developments forming a fifth area:

1. What structural mechanisms underpin picornavirus cell attachment/entry/uncoating/assembly?
To answer this we will determine new structures of viruses, uncoating intermediates and use scattering and imaging techniques to obtain information of dynamic states.
We will also try to understand the structure of the enveloped hepatitis A virion.
This will be the largest area of activity.

2. What is the structure of the Hepatitis B sAg particle?
To answer this we will investigate both XFEL and EM as methods to obtain high resolution information.

3. What is the molecular basis of membrane fusion in archaeal pleomorphic viruses?
To answer this we will use in vitro and in vivo methods, along with electron tomography and X-ray crystallography.

4. What is the molecular basis of genome packaging and transcription in viruses of the Reoviridae?
To answer this we will use novel EM analysis methods, plus X-ray crystallography.

Impact will arise in several ways.

Firstly our work will inform fundamental virology, providing structural paradigms that explain function.

Secondly it will provide new methods, protocols and reagents that may be of general value to structural biologists.

Thirdly it will inform commercial activity, in particular in (i) the area of small molecules and biologicals with antiviral activity and (ii) the area of novel or improved vaccines.

For instance there are at present no licensed anit-virals targeting picronaviruses, despite the serious impact on human and animal health that this family of viruses cause. A number of years ago a new class of anti-virals, capsid stabilisers were proposed as potential drugs, initially by the Rossmann laboratory (Purdue University). At that time despite some clinical efficacy development was not taken forward. More recently Biota have re-opened this line of investigation and are in phase 2 trials for a related compound, targeting those patients at greater risk from rhinovirus infection. The potential of such an approach for other picornaviruses has been illustrated by our recent work on compounds targeting EV71. We have used structure to guide the design of a highly potent inhibitor, which is patented and has undergone successful animal tests in China. We expect tests in humans to start this calendar year. We are also in discussion with SeaLIfe Pharma to investigate the exploitation of the compound for other picornaviruses. Whilst there is still a considerable gap between such early stage success and a useful drug, we believe that it should be feasible, ultimately, to provide a relatively small number of compounds which will be available in the event of a threat from an emerging enterovirus. We will also be alert to other therapeutic opportunities, and will, as appropriate, provide information about enzymatic targets (for instance we have recently determined [unpublished] the structure of a protease from the more clinically serious rhinovirus C). Compounds such as the capsid stabilisers mentioned above have additional potential value as excipients for vaccine formulation and perhaps also during antigen purification. We are investigating this, for instance, in Gates supported work on synthetic poliovirus vaccines. Indeed our work has a number of potential impacts on vaccines. For example by structure based capsid engineering we have improved the stability of some foot-and-mouth disease VLPs. We are using related methods to attempt to re-engineer enterovirus capsids, again with the aim of enabling a switch to non-virus based vaccines which would be much safer, and potentially more effective. This work is partly in conjunction with academic partners, and where appropriate, with commercial partners (such as MSD Animal Health). We anticipate further viruses being brought within scope for structure-based engineering during the next quinquennium.

The final area of impact for this research programme will be in the training and career development of a cohort of young research scientists. I have been able to work with very talented, energetic, enthusiastic scientists, some experienced, others at an early career stage. Of the order of a dozen of these have since gone on to leadership positions, in academia and industry. I place considerable importance on this aspect and aim to provide a good training environment.