Innate immune restriction of enveloped virus exit

Viruses that infect the respiratory tract are responsible for a huge number of global deaths every year, especially in vulnerable populations like the malnourished, children and the elderly. Two of the most significant respiratory viruses are RSV (respiratory syncytial virus) and MeV (measles virus). There is no vaccine to RSV and most children will have had this infection by the time they are 2 years old. Unfortunately, around 150,000 children a year die from this disease, mostly because the virus causes pneumonia and other serious infections of the airways. The situation with MeV is more complex because although we have a vaccine it has not been used to fully eradicate the virus. As a result, and because MeV itself is incredibly infectious, around 115,000 people still die from this infection every year. RSV and MeV are therefore important viruses to study in the laboratory and hospital setting. If we can improve our understanding of these viruses we can design better drugs and vaccines to tackle these infections, reducing the strain they place on global healthcare.

My research is focused on the immune response to RSV and MeV, especially during the early stages of disease when these viruses are establishing themselves in the human airway. This project is designed to answer medically and scientifically relevant questions such as:
1. How does the cell work to fight RSV and MeV infection, in particular when and where are these pathogens targeted for restriction?
2. How and why do these viral infections cause disease?
3. Why some people are more susceptible than others to these infections?
4. Whether the immune response itself can sometimes cause problems?

My project will focus on the early acting innate immune system. This is made up of a network of sensors and effectors that have evolved to detect pathogens, like viruses, and inhibit their replication and continued spread. I will examine how RSV and MeV are inhibited by this pathway, focusing on the formation and release of virus particles. This is an important stage in the viral life cycle because it is essential for continued infection. As such, improving our knowledge of this process could lead to the development of targeted antivirals.

To be more specific about the experiments we will perform: RSV and MeV steal their envelope, or coat, from the infected cell during a process called budding. This process is reliant on a number of viral proteins that work together to make infectious particles. I have developed high-throughput assays that let me investigate these proteins and the various stages of the viral life-cycle in greater detail. Using these assays I will examine whether individual proteins from the innate immune system can inhibit the formation of new virus particles. Separately I have developed new techniques that allow purification of the cellular membranes where RSV and MeV replicate. I will use this technology to examine what immune proteins are drawn into this fraction during infection. The proteins that I identify will then be characterised to examine how, where and when they work to inhibit these infections. In addition I will investigate their relevance in models of the respiratory tract and also examine whether genetic variation in these proteins affects their function.

To summarise; this project will examine and characterise the immune response to RSV and MeV, two important childhood disease of the respiratory tract.

Respiratory syncytial virus (RSV) and measles virus (MeV) are significant causes of human mortality, especially among children and the elderly. There is no vaccine for RSV and no licenced antivirals to tackle either virus. My research focuses on the innate immune response to these pathogens, particularly factors that inhibit viral egress. RSV and MeV are both enveloped paramyxoviruses whose exit is coordinated by viral matrix and glyco- proteins. In this project I will apply genetic screens and proteomics to identify inhibitors of these viral proteins and pathways.
My first approach combines quantitative assays for viral cell fusion and budding with a human interferon stimulated gene (ISG) library to identify inhibitors. These platforms, assays and approaches have been established in my laboratory and preliminary experiments, presented in my case for support, demonstrate their viability. The second approach is deliberately non-biased - in this system the membrane fraction from infected cells is compared, using quantitative proteomics (SILAC), to uninfected cell fractions to identify enrichment of immune-related proteins. This is relevant because RSV/MeV egress is continually membrane-associated, starting with viral protein trafficking through the ER, Golgi and endosomes and ending with plasma membrane-based budding or fusion events. Again, I have demonstrated the viability of this approach in preliminary experiments which successfully enriched the entire MeV virome. Finally, proteins or pathways that I identify and validate as inhibitors of egress will be broadly characterised to dissect their mechanism of action within the infected cell. This will be performed using an established biochemical and cell-biology based toolbox. Again I provide compelling information in my Case for Support to back-up their application. To summarise this is a cutting-edge application that will improve our understanding of the innate immune response to two globally relevant infections.

This proposal focuses on improving our knowledge of the innate immune responses to RSV and MeV, two important pathogens of humans. This will be performed through application of robust high-throughput assays, next generation quantitative proteomics and in-depth molecular biology. I anticipate the impacts from this project will be as follows:

Advancement of scientific knowledge within both the academic community and general public: The academic community will benefit directly from this research through advancement of innate immunity and RSV/MeV fields and the emergence of new areas for research. The field of paramyxovirus research is relatively small in the UK and establishing new links and connections between this area and that of viral innate immunity (which is altogether much larger) will impact greatly on future research and my own collaborations. I am committed to the dissemination of our research findings to the general public and through ongoing interactions with the ThinkTank science museum in Birmingham and separately my lab's blog (http://uobchv.blogspot.co.uk) we will publicise our research.

Development and showcasing of novel technologies and assay platforms: I have developed a number of novel strategies for integrating high throughout fusion and budding assays with genetic screens as well as quantitative, sub-cellular fractionation-based proteomics. By publishing and presenting these approaches I will foster their application in academia, industry and healthcare.

The provision of skilled people to the workforce: The PDRA and RT employed as part of this project will gain specific and transferable skills key to the future sustainability and development of the UK's scientific workforce including academic, management and technical skills which could be applied in the future (within or outside the scientific sector).

Future therapeutics against RSV and MeV: My research could be used to develop therapeutics to target RSV or MeV egress. There is certainly scope for developing antivirals, as currently there are no direct-acting drugs available to combat these infections, despite interest from pharmaceutical companies. Additionally, since my assays are designed to be used at high levels of throughput they could easily be optimised for drug screens. Antivirals of this nature would impact on health and wellbeing particularly amongst patients (especially children and the elderly) since RSV and MeV represent ongoing concerns for public health, both in the UK and globally. Of note, this approach could also lead to the development of broadly anti-paramyxoviral drugs as the mechanisms of egress may be conserved.

Future improvement of vaccine/recombinant virus production within the pharmaceutical and biotech industry: There are also broad and important implications for my research in industry since the efficient production of viral glycoproteins is the basis for many recombinant vaccines and pseudotyped viral therapeutics. Uncovering innate immune mechanisms that restrict production could therefore impact on the design of higher yield systems with more efficient production.