Functional investigation of Epstein-Barr virus infection and latent gene function using natural variants found in normal infections and cancer cells

Epstein-Barr virus (EBV) contributes to several types of cancer including Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma, post-transplant lymphoproliferative disease, nasopharyngeal carcinoma and gastric carcinoma. It also causes most cases of infectious mononucleosis (glandular fever). In recent years there has been major progress in developing early diagnosis of some EBV associated cancers and clinical trials are in progress for improved immunotherapy. The US National Cancer Institute is also supporting development of an EBV vaccine.

There is increasing evidence that natural variation in the EBV genome DNA sequence can affect the frequency or severity of disease, and affects normal EBV biology and infections.

We shall investigate two major aspects of EBV genome variation that will underpin making improved therapies and strategies for prevention of disease more widely effective:

First, the largest natural variation of EBV divides virus strains into two types, called type 1 and type 2, which leads to striking differences in virus biology. They have different geographic distributions, with type 1 (which is more diverse) occuring worldwide but type 2 having its highest incidence in sub-Saharan Africa and other parts of the world where other infectious diseases are very intense. We aim to understand the mechanisms that underlie the differences in biology of these virus types, and how they might differently contribute to disease. In particular we will investigate how different types of EBNA2 and EBNA3 proteins work together to manipulate the infected cell and examine whether proteins on the virus surface (that are the vaccine candidates) alter what cells EBV can infect.

Secondly, we shall investigate the significance of diversity of the EBNA1 gene. EBNA1 is the only EBV protein made in all EBV associated cancers, and is essential to EBV persisting in those cancers, so it is an attractive target for therapy. The differences between EBNA1 protein sequences may affect its contribution to cancer and response to potential EBV drugs. We have identified the main variants of EBNA1 and will now be able to will test the effects of these widespread, naturally occurring variations to ensure virus diversity is factored into efforts to provide new therapies and immunisations, and to understand the biology of EBV associated diseases.

Epstein-Barr virus (EBV) contributes to several types of cancer and causes infectious mononucleosis. There is increasing evidence that natural variation in the EBV genome is important for disease pathogenesis and impacts on normal EBV biology. We have accumulated over 250 EBV genome sequences and will now focus on the consequences of the viral sequence variation for EBV-associated disease, biology and therapy. We shall
- Use our new resources to re-evaluate the biology of type 1 and type 2 EBV, following the discovery that type 2 EBV infects T cells (in addition to B cells)
- Test the significance of variation in EBNA1, the protein that controls maintenance replication of the viral genome but also has many other activities potentially relevant to cancer

Specifically, we shall
1. Use our newly generated recombinant type 2 EBV bacterial artificial chromosome (BAC) to produce virus and investigate the ability of type 2 EBV to infect T cells. We shall investigate the effects on the T cell, and distinguish the contributions of EBV glycoprotein variants and EBV nuclear proteins (EBNAs) linked to type 1 and type 2 on B and T cell infection.
2. Test our hypothesis that type 2 EBV may be better at achieving infection and outgrowth of activated lymphocytes than type 1 EBV, potentially consistent with its higher prevalence in populations with chronic immune activation caused by other infectious diseases (eg Malaria).
3. Functionally investigate the very close type-specific linkage of EBNA2 and EBNA3s. We propose there must be some novel functional compatibility that explains why strains almost always have type 1 EBNA2 with type 1 EBNA3 or type 2 EBNA2 with type 2 EBNA3s.
4. Determine the functional impact of variation in EBNA1 that we have identified. We will examine the co-operative DNA binding, oligomerisation and anti-EBNA1 drug susceptibility. We shall also test the impact of variation on known EBNA1 functions that might contribute to EBV associated cancers

Impact Summary

The main impacts will be achieved by
1. Clarifying the different infection tropisms of the main types of EBV, the role of gp350 and gp42 in tropism/ infection efficiency and the relationship of tropism/ infection efficiency to chronic immune activation. This has implications for EBV associated diseases with Africa and other areas of high incidence of infectious diseases.

2. Testing the significance of gp350 and gp42 variation will affect vaccine design for EBV

3. The novel mechanisms of interaction of EBNA2 and EBNA3 we aim to investigate may suggest new therapeutic approaches for EBV associated diseases expressing those proteins, such as post-transplant lymphoproliferative disease.

4. Identifying variants of EBNA1 that are more likely to contribute to EBV associated cancer may lead to risk profiling of EBV infection.

Who will benefit?
The increased academic knowledge and improved understanding achieved in this project will benefit academic researchers, clinicians and pharmaceutical companies active in this research area. Eventually the results will help patients suffering from diseases associated with EBV.