Epstein-Barr virus (EBV) is a common herpes virus that establishes life long persistence in the human host trough an elaborate regulation of different latency types and can directly transform B- lymphocytes. EBV causes the infectious mononucleosis and associates with several human malignancies such as Burkitt's lymphoma (BL), Hodgkin's disease, AIDS-associated B-cell lymphoma, primary CNS non-Hodgkin's lymphoma, gastric adenocarcinoma, X-linked lymphoproliferative syndrome, nasopharyngeal carcinoma and post transplant lymphoproliferative disease. In vitro EBV transforms B-cells into lymphoblastoid cell lines (LCLs). EBV uses the normal biology of B-lymphocytes to drive B cell development and survival in the absence of normal B- cell receptor (BCR) signals.
The general objective of the project is to define the mechanisms by which EBV regulates B cell fate. Specific objectives are:
1. To investigate the involvement of EBV-encoded proteins in modulation of receptor- and drug-induced apoptosis;
2. To test the hypothesis that EBV-encoded latent proteins regulate the expression of the cell surface receptor CD150;
3. To analyze the signal transduction via CD150;
4. To study protein-protein interactions of EBV-encoded proteins with the components of signal transduction pathways.
Specific tasks of this project will be:
T1. To obtain and characterize B cell lines infected by EBV and transfected by EBV-encoded proteins. This will include development of an experimental systems based on B cell lines, and characterization of cell surface phenotype and expression of apoptosis-associated proteins in these cell lines;
T2. To elucidate the involvement of a distinct EBV-encoded proteins in modulation of apoptosis. This will entail studies of apoptosis initiated via cell surface receptors and drug-induced apoptosis;
T3. To define the signal transduction pathways and mechanisms by which CD150 regulates immune responses. The signal transduction pathways initiated via CD150 will be investigated and the roles of the adaptor protein SH2D1A, the tyrosine kinases Lyn and Btk, the phosphatases SHIP and SHP-2 in the regulation of CD150-initiated signal transduction pathways will be clarified;
T4. Interactions between EBV-encoded and cellular proteins will be studied using yeast 2-hybrid system and surface plasmon resonance;
T5. Modelling of CD150 signal transduction pathways in B-cells using generalized chemical kinetic simulation method. A wide range of biochemical, molecular biology, immunological, confocal microscopy, immunocytochemistry, Fluorescence-activated cell sorting, functional (including cell death studies), surface plasmon resonance technique, computational and mathematical methods will be applied. Potential results will be expected: Experimental model systems will be created. Involvement of EBV-encoded proteins in modulation of apoptosis will be clarified. The signal transduction pathways that are initiated via CD150 will be determined and the role of CD150-associated proteins in these pathways will be found. Identification of the cellular components that are capable to interact with EBV-encoded proteins will allow creating a mathematical and computer model how EBV-encoded proteins could affect signalling pathways in B lymphocytes and regulate B cell fate. This will help to find cellular signal transduction pathways that are targeted by EBV and design the strategy of anti-viral therapy.