Structural studies of viruses and their interactions with cells

Viral diseases have been responsible for massively damaging pandemics in the past, the best known example being the influenza outbreak of 1918. In recent years we have mercifully escaped such global disasters, however there is still a real and present risk. Thus the September 2019 report of the WHO Global Preparedness Monitoring Board (https://apps.who.int/gpmb/assets/annual_report/GPMB_annualreport_2019.pdf) warned that "there is a very real threat of a rapidly moving, highly lethal pandemic of a respiratory pathogen killing 50 to 80 million people and wiping out nearly 5% of the world's economy". One of their required actions is "ensure adequate investment in development of innovative vaccine and therapeutics, surge manufacturing capacity, broad-spectrum antivirals and appropriate non-pharmaceutical interventions". The programme proposed here does not primarily address lethal respiratory viruses, however it does address two important families of viral pathogens, the Picornaviridae and the Reoviridae.

Picornaviruses are responsible not only for a very large proportion of respiratory tract infections, but also for other major epidemics, notably several million cases per year of hand-foot-and-mouth disease. Although one of the best known picornavirus diseases, polio, is almost eliminated there has been a concerning increase in cases recently (https://www.who.int/news-room/detail/07-01-2020-statement-o-the-twenty-third-ihr-emergency-committee-regarding-the-international-spread-of-poliovirus) and there are still serious cases of acute flaccid paralysis due to another enteroviruses. Frequent recombination events occurring between two co-infecting picornaviruses will no doubt lead to the emergence and re-emergence of numerous picornavirus caused diseases. Similarly, amongst the Reoviridae rotaviruses are estimated to cause several hundred million cases of gastroenteritis every year. Thus the viruses we propose to target represent significant global health burdens, but there are still a rather small number of vaccines available and no licenced anti-picornavirus drugs. There is a clear unmet need for novel therapeutics.

Due to the continuing revolution in cryo-electron imaging it is now a golden age for structural virology and yet there remain major gaps in our understanding of how they work. The idea driving the current programme is that integrated structural biology is now sufficiently powerful that it can provide a wealth of atomic level information to feed into the search for new therapies, but can also contribute to the broader cellular level understanding of the full complexity of the virus life cycle. The goal of this programme is to make genuine contributions to answering some fundamental questions, and to at least set a direction of travel towards improved treatment.

The proposal targets RNA viruses, especially the Picornaviridae, small icosahedral viruses (30nm diameter) with a +ve single stranded genome, and the Reoviridae, larger icosahedral viruses (~80nm across) with 10-12 double stranded genome segments which enter cells as intact cores forming transcription factories in the cytoplasm. Picornaviruses are responsible for a large proportion of respiratory tract infections, and also for other major epidemics, notably several million cases per year of hand-foot-and-mouth disease. Similarly, amongst the Reoviridae rotaviruses are estimated to cause several hundred million cases of gastroenteritis every year. New therapeutics are needed since there are only a rather small number of vaccines available and no licenced anti-picornavirus drugs.

We propose to take advantage of, and progress, the rapid technological developments in cryo-imaging of biological samples, especially with electrons, to make a coherent programme for the integrated investigation of these viruses. In particular we will use electron tomography to visualise viruses within cells in close to atomic detail. Since the workflows are complex the duration and size of the programme award requested is essential, and there will be real synergy between the different elements of the programme, increasing productivity and impact.

The programme has four aims. The first three address gaps in basic knowledge that can be filled by integrated structural studies. The fourth links fundamental knowledge to translation, including pre-competitive research into anti-virals:

Aim 1 - Improve structural understanding of Reoviridae replication
Aim 2 - Improve structural understanding of how picornaviruses enter cells and replicate
Aim 3 - Improve structural understanding of immune responses
Aim 4 - Use structure to guide anti-viral inhibitor design for drug and vaccine therapy

Academic impact
The impact on Academic Beneficiaries is discussed in that section. Essentially the results will have impact on the development of fundamental knowledge and so will alter the course of research. In addition the methods will have a broader applicability beyond structural virology to include broader structural biology and ultimately, if successful, to impact the practice of cell biology. I believe that the use of rigorous structural biology methods in cell biology will ultimately help improve quantification and reproducibility.

Therapeutic impact
I have briefly described elsewhere why there is a pressing need for new vaccines and anti-viral drugs against some of the viruses that we propose to study. In general there is a very substantial time-lag before basic science makes a real impact in the clinic and so it can be hard to trace direct connections. However looking back over the work I have been involved in it seems reasonable to infer that the structural studies of HIV reverse transcriptase inhibitors that we were involved in have had an impact and helped the development of a generation of NNRTI drugs. Similarly structural vaccinology, which we have been proponents of for some years now seems to be reaching the point where it is affecting products which are coming to market (notably our foot-and-mouth disease VLP work with a consortium led by Bryan Charleston of the Pirbright Institute, which is being scaled up by MSD Animal Health at the moment). As an example pertinent to the present application, I am pleased to see that the crystal system we developed which led to us discovering the mode of action of certain compounds with activity against Ebola virus has now been taken up by others (see PDB deposition 6NAE) and used to investigate a further chemical class of compounds binding at that site. Similarly a picomolar inhibitor we designed to inhibition's hand-foot-and-mouth disease viruses has been taken forward in China. There is a large gap from this discovery phase to compounds in the clinic, but such precompetitive work is essential if we are to have any hope of preparedness for future outbreaks.

Career development
The career development and training of young researchers will be another area of impact. I believe that Oxford in general and Strubi and my laboratory in particular provide a good environment for young scientists and we are able to attract very talented people. Over the years students and young postdocs have mostly developed a successful career in science, sometimes academic, sometimes in industry. An example of the latter is Harren Johti, who worked as a PDRA for me, later was involved in setting up Astex Pharmaceuticals and has recently been elected to the Royal Society. It is perhaps invidious to select one individual, I could equally well have chosen an excellent student who has since established another successful drug discovery company, or any one of a large number of students who have now gone on to be successful group leaders in academia in the UK and around the world. It is one of the delights of science to be able to give talented people the chance to develop.