Molecular and cellular mechanisms of synapse-mediated spread of Epstein Barr virus: overcoming the CD21-restricted cellular tropism.

Epstein Barr Virus (EBV) is a common, orally-transmitted virus, infecting most of the human population. EBV maintains a silent infection for the lifetime of the host by hiding in a subset of lymphocytes called B cells. EBV infection of B cells in the laboratory has been extensively studied. In the laboratory, infected B cells are efficiently transformed into continuously growing cells with the potential to cause cancer. This happens only rarely in the body, but when it does can cause B cell cancers including Hodgkin lymphoma. One reason why B cell cancers are so rare is because infection is kept under control by immune responses involving other cells, including T and natural killer (NK) cells.
The mechanisms of EBV entry into B cells have been extensively studied. Proteins on the virus (gp350 and gp42) interact with virus receptors on the cell surface (CD21, HLA class II respectively) to bind the EBV to the B cell. Thereafter further virus proteins (gp82, gp25) trigger virus internalisation. However, infection of other cell types, including epithelial, T and NK cells that do not express the CD21 are also observed. These infections are associated with either virus transmission to a new susceptible host (epithelial), or are associated with diseases and malignancies of epithelial, T and NK cells.
Epithelial cells line the oral cavity (oropharynx) and create a barrier for entry of EBV into an uninfected host, and for escape of EBV from an infected host for transmission to a new susceptible host. Epithelial cells are thought to be the major site of EBV replication in the oropharynx; however they do not express CD21, and EBV infection of epithelial cells in the laboratory is poor. I have previously shown that infection of epithelial cells can be greatly enhanced by first binding the virus to B cells. EBV binding to CD21 triggers adhesion molecules and EBV on the B cell surface to polarise then 'adhere' to the epithelial cells, bringing EBV into close contact with epithelial cells and enabling efficient infection. This process is probably similar to the situation in the oropharynx, where epithelial cells and lymphocytes, including B cells, are in close contact with each other.
In our model of EBV infection of epithelial cells, we suggest EBV on the B cells may come from fully matured B cells, called plasma cells, which when infected with EBV produce new infectious virus. This EBV may be released from the plasma cell to interact with the resting B cell as above. Or, as increasingly observed with other viruses that infect lymphocytes (like HIV), the plasma cell may interact directly with the epithelial cell, cause EBV inside the plasma cell to relocate to the point of cell-to-cell contact and transmit directly from the plasma cell to the epithelial cell, thereby evading anti-EBV antibodies. Here, I intend to extend my studies to understand how EBV can infect epithelial cells using plasma cells as the virus donor, and determine the molecular requirements of epithelial cells and EBV for efficient infection, so we can understand how to disrupt EBV transmission with vaccines.
EBV associated T and NK cell diseases range from illnesses that appear to be a consequence of an over-active immune system (EBV-associated haemophagocytic lymphohistiocytosis, chronic active EBV), to aggressive malignancies (extranodal T/NK cell lymphoma, aggressive NK cell leukaemia).T and NK cells do not express CD21 and we do not understand how these cells can be infected with EBV. Using two different methods, I aim to determine how infection of these cells is achieved: (1) Using plasma cell interaction with T and NK cells, and (2) infecting CD21-positive immature T and NK cells. With this new information, we will be able to develop ideas of how the virus may be causing the T and NK cell diseases, and begin develop new treatments for these treatment-resistant diseases.

The aim of this research is to understand one of the fundamental aspects of viral pathogenesis: How does Epstein Barr virus (EBV) gain entry to epithelial cells, T and NK cells which do not express CD21, the virus receptor?
(1) Efficient infection of epithelial cells requires EBV-loaded B cells to act as virus donors; physiologically this is likely to be lytic plasma cells. Using lytic B cell lines at different stages of plasma cell differentiation, or non-transformed B cells induced into plasma cell differentiation by CpG DNA and interferon alpha treatment, we will examine the formation of a virological synapse between plasma cells and uninfected epithelial cells. Viral synapse formation is triggered by viral and cellular molecules which polarise with adhesion molecules to the point of cell-cell contact. Using fluorescently-labelled recombinant viruses and cellular components, we will monitor relocalisation of virus capsid, adhesion molecules and intracellular transport machinery by live cell confocal microscopy and determine their individual roles in synapse formation.
(2) T and NK cells are refractory to EBV infection in vitro so may require a donor B cell for infection. Lytic plasma cells will be cultured with a panel of well characterised, HLA-matched CD4 and CD8 T cells and interferon alpha-induced NK cells to examine transmission of EBV via the virological synapse. Using a panel of recombinant viruses, we will determine the viral and cellular factors required. Finally, CD21 is temporally expressed on immature T and NK cells during differentiation from lineage negative haematopoietic stem cells into T or NK cells. Using the OP9-DL1 system to in vitro differentiate T and NK cells and using we'll take advantage of this temporal CD21 expression and infect the immature cells prior to lineage commitment. We will then be in a position to determine if EBV infection per se is sufficient to drive the T and NK associated pathologies.

As a pathogenic, oncogenic virus, Epstein Barr Virus is the subject of much research worldwide. However, despite this there is a great deal of uncertaintly surrounding the mechanisms by which it infects non-B cells, namely epithelial cells, T and NK cells. This is relevant not only in the study of cancer, but, at a more basic level, in our understanding of how the virus is shed from epithelial cells into the saliva to infect the next host. This is particularly relevant not just to understand the basic biology of the virus, but also in the investigation of EBV-associated malignancies such as nasopharyngeal carcinoma and NK/T cell lymphoma. The identification of the mechanisms by which EBV enters cells not expressing the virus receptor, namely epithelial cells and particularly T and NK cells will (1) increase the academic knowledge base in the UK. (ii) Will be of interest to biotechnology companies and academic groups involved in generating anti-herpesvirus vaccines. This study will indicate what virus proteins are essential in transmission and indicate whether, during cell-cell transmission the virus can be accessible to the neutralising antibodies. (iii) Will be of interest to clinicians and scientists working on EBV infection of T and NK cells. Nothing is currently known of how the virus enters T and NK cells, so equally little is known of the pathogenic mechanisms by which EBV causes disease in these cells. Furthermore, malignancies associated with EBV in T and NK cells are highly resistant to conventional treatment and the only model we currently have to examine EBV in these cells are cell lines, therefore reliable infection of primary cells will also be of interest to biotechnology companies in the development of improved treatment. (iv) Will be of interest to charities, such as one set up by the family of a patient who died from EBV-associated NK/T cell lymphoma.
Given the potential interest of biotechnology companies in development of new treatments, and in vaccine developemnt, this could be an advantage economically to the UK.
Diagnosis of EBV-associated disease in non-B cells is devastating, not least because the prognosis is invariably poor. Therefore, in the future this may benefit people who have EBV-associated cancers that are refractory to current treatments. This is particularly applicable outside the UK given the worldwideincidence of nasopharyngeal carcinoma, haemophagocytic lymphohistiocytosis, chronic active EBV, extranodal NK/T cell lymphoma etc.
The project will provide valuable experience in state of the art microscopic techniques, particularly real time confocal microscopy. It will also provide experience in many molecular biology and cell biology techniques which are transferable skills and can be taken to any laboratory environment.