Epstein-Barr Virus (EBV) has been implicated in the pathogenesis of AIDS-related lymphomas. B lymphocytes can be adapted to HIV infection in vitro following activation by EBV, suggesting that retrovirus coinfection may be operative in B cell tumorigenesis. Activation of the autocrine loop, whereby a cell both secretes and responds to soluble growth factors can provide the autonomy necessary for malignant proliferation and has been implicated in the continuous growth of Epstein-Barr virus (EBV) infected B lymphocytes. One B cell differentiation antigen (CD23), is superinduced on B cells by EBV infection. A shed form of CD23 has been implicated as the B cell growth factor found in culture medium from these EBV infected lymphoblastoid cell lines (LCL). Using biochemical and recombinant DNA techniques I propose to investigate the effects of HIV, EBV infection or EBNA-2 expression on induction of CD23 expression in an EBV-negative continuous Burkitt tumor cell line (Louckes). S1 nuclease protection studies will show which form(s) of CD23 (A or B) mRNA is induced. Studies will be performed to determine if phosphorylation of CD23A (Tyr-Ser) or CD23 (Ser) occurs. I will examine the molecular nature of the CD23 dimer that is seen following virus infection and will determine if the rate of surface shedding of 125 I-CD23 is altered. I also propose to purify soluble CD23 (sCD23) from LCLs and recombinant CD23 (rCD23) from transfected cell lines (GH3 and Rat-1) to assess their autocrine growth promoting activity by 3H-thymidine incorporation in antigen/mitogen activated B cells and starved LCL's. The surface protein through which CD23 (or an associated 12 kDa molecule) binds and mediates its biological responses will be characterized by both 125 I-CD23 receptor binding analysis and heterobifunctional chemical cross-linking. Using the eukaryotic expression vector (pCMVIE.AKR), intact CD23 will also be transfected into GH3 and Rat-1 cells to study the mechanisms of receptor cleavage. Similar studies will be performed on CD23 purified from LCL or transfected cells that is incorporated into phospholipid vesicles. Intact CD23 will also be transfected into B lymphocytes or an EBV-negative Burkitt's lymphoma cells to study whether expression of CD23 causes phenotypic changes associated with transformation. Lastly, mutant CD23 molecules will be constructed in order to delineate the mechanism of receptor shedding and to determine the active domain(s) involved in cell binding and cell activation.
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| Bagley, J; Dillon, P J; Rosen, C et al. (1994) Structural characterization of broadly neutralizing human monoclonal antibodies against the CD4 binding site of HIV-1 gp120. Mol Immunol 31:1149-60 |
| Marasco, W A; Haseltine, W A; Chen, S Y (1993) Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A 90:7889-93 |
| Marasco, W A; Bagley, J; Zani, C et al. (1992) Characterization of the cDNA of a broadly reactive neutralizing human anti-gp120 monoclonal antibody. J Clin Invest 90:1467-78 |
