's abstract): Epstein-Barr virus is a pathogenic human herpesvirus that persists for the lifetime of the host. Recently, resting memory B cells, that express the viral latent gene LMP 2, have been identified as a site of persistence. How these cells are produced and maintained is unknown. This stands in stark contrast to the behavior of the virus in vitro where it is known to be a powerful immortalizing virus encoding at least nine latent proteins that include known growth promoting genes and oncogenes. The hypothesis is that EBV-infected B cells are maintained like normal B cells with the resting cells circulating in the periphery and the proliferating blasts being retained within the lymphoid tissue. This implies that the numbers of circulating cells must be replenished through proliferation in lymphoid tissue. The goals of this study are to understand: 1. How EBV can affect the transitions from a proliferating to a resting cell and thereby gain access to and persist in the memory B cell compartment. 2. How and via which latency states the infected memory B cells replace and maintain their numbers. 3. How this system is perturbed in immunosuppressed individuals. To achieve this goal the applicant will: 1. Identify the infected cell types found in mucosal lymphoid tissue and arising in the periphery of immunosuppressed patients using DNA PCR to identify the virus and monoclonal antibodies that distinguish discrete populations of mature B cells. 2. Use monoclonal antibodies, magnetic beads and the FACS to isolate the different populations of infected cells and test them for latent gene expression using RT PCR and for viral replication using Gardella gels. 3. Test if different forms of latent infection (latency 0,1,2 and 3 for example) can give rise to each other by tracing infected cells in vivo using a marker of clonal relatedness and by observing the effects of cellular signaling on the state of viral latency. 4. Use mouse model systems to study how LMP2 positive cells may escape immunosurveillance (LMP2 transgenics), and identify the potentially oncogenic populations in immunosuppressed donors (SCID mice).
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