Functional analysis of the Epstein Barr virus nuclear antigen leader protein (EBNA-LP) in a viral context.

Epstein-Barr virus (EBV) infects most of the human population. Infection normally has no symptoms but some people develop infectious mononucleosis (glandular fever) when they become infected. The virus persists life-long in a dormant (latent) state in certain white blood cells. However, occasionally EBV causes cancers of the white blood cells, particularly where individuals are also infected by HIV or Malaria. EBV is also associated with a cancer of the nasopharynx which is unusually common in Southern China. In all, about 1% of all cancers worldwide are thought to involve EBV. The process of establishing EBV latency is governed by some of the viral genes, including the EBV nuclear antigens (EBNAs). The focus of this research grant is one of the EBNAs, EBNA-LP. Previous research has shown that EBNA-LP is important for the ability of EBV to change B cells into a more cancer-like state (transformation): in cell culture EBV can 'transform' B cells to divide indefinitely. EBNA-LP has also been shown to bind to a number of cellular proteins, and to enhance the abilities of another EBNA, EBNA2, to activate certain genes. However, there is no clear understanding of what EBNA-LP does in the context of virus infection. We also don't know whether any of the reported interactions of EBNA-LP with cell proteins are relevant to its functions in EBV infection.
To overcome this, I have developed new methods to create EBV strains in which EBNA-LP has been deleted or engineered to contain defined mutations. This was an exceptional technical challenge due to the complex nature of the EBNA-LP gene. Using these viruses, we will be able to assess what roles are played by EBNA-LP in the transformation of B cells, and to identify genes that are controlled by EBNA-LP. I will also clarify the ability of EBNA-LP to interact with cellular proteins, identifying which parts of EBNA-LP bind to these proteins. By correlating the ability of EBNA-LP mutants to interact with a specific host protein and specific defects in transformation, I will identify the biological significance of these interactions and elucidate which ones may provide novel therapeutic targets for EBV. The protein interfaces involved in the interactions and the sites of protein modification may provide targets for chemical compounds that can inhibit those interactions. Revealing precisely what EBNA-LP does for the virus and its mechanism will thus allow future development of assays for novel anti-EBV therapies that may allow elimination of the virus or novel treatments of EBV associated cancers. There are many examples in cancer biology and immune evasion where key cell mechanisms have been discovered by investigation of virus proteins that have evolved to interfere with cell processes. It is therefore hoped that more general insights into disease mechanisms will also come out of the experiments in this research proposal.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is involved in 1% of cancer incidence worldwide. In vitro EBV transforms resting B cells into lymphocyte cell lines. EBNA-LP is one of the EBV nuclear antigens (EBNAs) important for this process. Exploration of its role in EBV biology has been neglected because of its complex repetitive nature and the technical challenge of making mutants of EBNA-LP in EBV. I propose to take a genetic approach to clarify the role of EBNA-LP in B cell transformation and identify the mechanisms central to its key biological properties.

In vitro EBNA-LP is able to enhance the ability of EBNA2 to activate gene transcription. Sp100, NCoR and HDAC4 have variously been implicated as mediators of this effect. Using an EBNA-LP-knockout EBV (LPKO) and its revertant, we will identify the functions of EBNA-LP in B cell transformation. We will analyse the phenotype of LPKO-infected B cells, and undertake a microarray analysis of LPKO-infected BL31 cells to allow us to identify genes that are co-regulated with EBNA2 and with the EBNA3s, as well as those altered independently by EBNA-LP. In parallel we will map the interaction domains of 10 known EBNA-LP-binding partners. Having identified EBNA-LP mutants that lack defined interactions, we will complement LPKO EBV to establish which mutants restore which phenotypes. This will identify candidate mediators of those phenotypes, which can be tested by knockdown or overexpression. We will establish whether enhancement of EBNA2 function plays a significant role in gene regulation, by altering expression of Sp100, NCoR and HDAC4, and by analysing a recombinant EBV encoding S36 mutants of EBNA-LP. Finally we will use chromatin immunoprecipitation to reveal mechanisms of gene regulation by EBNA-LP and the proteins that mediate its effects. This program will integrate existing knowledge of EBNA-LP interactions with its biological role, providing mechanistic insights for future application.

Academic Impacts
This project aims to refine established observations and combine them with a new understanding of EBNA-LP function, thus enhancing the knowledge economy. The mutagenesis strategy and recombinant viruses represent research resources for future studies of EBNA-LP function. Additionally the project will give an opportunity for research training of the postdoc and PhD student to learn in an interdisciplinary team environment where we will benefitting from each others expertise in genomics and bioinformatics, protein chemistry, cell biology and synthetic biology, increasing the pool of scientists skilled in complex DNA manipulation. Further academic impacts are detailed in the Academic Beneficiaries section.

Economic Impact - Biotechnology industry.
EBV is used as a platform for monoclonal antibody discovery. There are potential discoveries to be made from this project that could be spun out to develop a novel system for monoclonal discovery and production. Monoclonal antibody development is an economically important therapeutic area with potential for a widespread impact on patient health and wellbeing. This is detailed in the Pathways to Impact document.

Societal impact - Education
Primary academic research is increasingly accessible to the general public, through iTunes U and increasingly specialist podcasts and blogs. I will seek to publicise any observations that are of interest to a non-specialist audience on an accessible platform as described in the Communications plan.

Societal Impact - Health and wellbeing
EBV infection is widespread in the world population, and has been causally implicated in lymphomas and carcinomas, and less robustly correlated with some autoimmune disorders. By taking studies of EBV biology progressively closer to physiologically relevant systems, we will be increasingly able to identify causal mechanisms and consequently devise cures for EBV-related disease, benefitting people suffering from such diseases. This project will enhance understanding of virus biology, and define potential targets for design of therapeutic approaches, as detailed in the Pathways to Impact statement.