Intracellular antibodies """"""""intrabodies"""""""" are powerful molecular tools that can be used to interfere with biological process inside the cell in a highly specific manner. In this five year competitive renewal application, we proposed to use our 15 billion member human single chain antibody (sFv) phage-display library to isolate panels of high affinity human anti-HIV-1 tat and anti-human Cyclin T1 intrabodies that can later be used as models and as discovery tools for small molecule inhibitors. These intrabodies will be functionally mapped for their ability to block Tat-mediated HIV-1 LT transactivation and HIV-1 replication in transduced CD8+ T-cell depleted PBMCs from uninfected and HIV-1 infected patients. We have developed high throughput screening procedures to accomplish these goals. Anti-tat and anti-hCycT1 sFv proteins produced in E. Coil will be used to determine antibody binding constants for Tat and hCycT1, and for further refinement in Tat and hCycT1 functional epitope mapping studies, respectively. We will concentrate on identifying anti-hCycT1 intrabodies that block binding of hCycT1 to Tat protein and TAR RNA but do not disrupt hCycT1-cdk9 interactions, since blocking the hCycT1-cdk9 protein:protein interaction could disrupt the normal functioning of the pTEFb complex which is required for transcriptional elongation of cellular genes. We will also evaluate several normal cellular functions in stably transduced CD4+ T-cells that express anti-hCycT1 intrabodies to determine if these functions are altered by the anti-hCycT1 intrabodies that exhibit anti-viral activity. The effects of anti-hCycT1 intrabodies on several cellular and viral promoters will also be examined. We will also use a bicistronic, tat-independent, self-inactivating (SIN) HIV-1 vector to transduce CD8+ T-cell depleted PBMCs from uninfected and HIV-1-infected patients to simultaneously express both anti-tat and anti-hCycT1 intrabodies that each alone has been demonstrated to have potent anti-viral activity. We will determine if the combination of these two intrabodies results in additive or synergistic inhibition of HIV-1 replication. We will also use anti-tat intrabodies to evaluate their ability to block the release of Tat protein from Tat-transfected and HIV-1-infected cells in an effort to delineate the mechanism(s) of non-classical secretion of Tat from HIV-1-infected cells. We will also examine the ability of extracellular anti-tat sFv proteins to inhibit the cellular uptake of Tat protein and the ability of these extracellular anti-tat sFv proteins to inhibit a spreading HIV-1-infection. These latter studies will serve to epitope map the region(s) of Tat protein that can be used to prevent the proposed extracellular modes of action of Tat in the pathogenesis of HIV-1-infection and AIDS. Collectively, these studies should provide valuable reagents that can be used to delineate critical functional epitopes on Tat and hCyc T1 that are involved in Tat-mediated transactivation, HIV-1 replication and HIV-1 pathogenesis. Our immediate goal is to use this knowledge to aid in small molecule inhibitor design, our long term goal is to use these intrabodies in a clinical gene therapy trial.
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