The long term objective of the proposed research is to define the essential contribution of the latent Epstein- Barr virus (EBV) nuclear protein EBNA-3A to EBV-induced proliferation of B lymphocytes. EBV's ability to induce proliferation of primary B lymphocytes is believed to be central to the establishment of long term latency, and thus to the development of EBV-associated malignancies that typically occur years after primary infection. EBV-associated malignancies continue to occur in HIV-infected patients despite the advent of HAART, and thus the mechansims by which EBV establishes latency, as well as its role in development of disease, warrant futher investigation. An important target of EBNA-3A in EBV-immortalized cells is Jkappa, through which EBNA-3A represses transcription. Our preliminary studies, as well as data from other labs, strongly support our hypothesis that EBNA-3A also has Jkappa-independent functions. Although both Jkappa-dependent and -independent functions of EBNA-3A are likely to play significant roles in EBV-induced cellular proliferation, there are significant gaps in our knowledge regarding these additional pathways and the downstream effects. To elucidate the functions of EBNA-3A that contribute to EBV-mediated proliferation, we propose three integrated aims to address these gaps in our knowledge. Using transcriptional profiling, we have identified cellular genes regulated by EBNA-3A that are likely to be biologically relevant to EBV-induced proliferation. Under Aim 1, we will identify the pathways through which EBNA-3A regulates these genes, as well as determine their biological relevance to continued proliferation of EBV-immortalized B-cells. Under Aim 2, we will identify proteins through which EBNA-3A mediates its effects in EBV-transformed B lymphocytes by purification of EBNA-3A containing protein complexes. The finding that the EBNA-3 proteins are expressed in a subset of Burkitt lymphomas forms the basis for Aim 3, where we will determine whether EBNA-3A increases the tumorigenicity of BL cells using a xenograft model and whether it affects tumor development in a mouse model of BL. These three aims, supported byShiv A. Prasad, Ph.D. [unreadable] Chief, Transplantation Immunobiology Branch [unreadable] Division of Allergy, Immunology, and Transplantation [unreadable] National Institute of Allergy and Infectious Diseases - NIH [unreadable] 6610 Rockledge Dr., Rm. 3035 [unreadable] Bethesda, MD 20892-6601 our preliminary studies, should greatly extend our understanding of the biochemical functions of EBNA-3A, as well as the downstream effectors mediating its essential contribution to EBV-mediated immortalization of B lymphocytes, and thus the pathogenic potential of this oncogenic herpesvirus. [unreadable] [unreadable] [unreadable]