Understanding the molecular mechanisms used by human immunodeficiency virus type 1 (HIV-1) to kill CD4+ T lymphocytes should critically contribute to finding new therapies capable of interrupting the progression to AIDS which follows HIV infection. Utilizing a transfection system previously developed by our laboratory employing Jurkat CD4+ T cell lines, we have defined a central biochemical mechanism by which HIV-1 kills CD4+ cells. We showed that HIV-1 induces a form of programmed cell death (PCD) triggered by tyrosine phosphorylation of cellular proteins, which leads to hyperphosphorylation of cyclin dependent kinase p34cdc2, an enzyme critically involved with the regulation and disregulation of the cell cycle at the G2/M checkpoint. We demonstrated that primary, clinical HIV-1 isolates use this mechanism ex vivo to kill CD4+ peripheral blood lymphocytes contributing to both the single cell and multicellular cytopathic effect commonly associated with pathogenic clinical variants of HIV-1. These studies employed antibodies to a G2/M phase-specific cell division cycle protein, cyclin B, to establish that cells dying of HIV cytopathicity were aberrantly arrested at the G2/M interface of the cell cycle. These antibodies can now be used to directly study T cells from HIV-infected individuals to learn 1) what population of T cells in infected individuals undergoes this killing process and 2) whether the percentage of affected cells can be used to predict progression to AIDS. We further demonstrated with a panel of metabolic inhibitors that HIV-mediated PCD substantially differed from the programmed death of CD4+ T cells which occurs during normal T cell development (negative selection). This finding has thereby increased the likelihood that clinically useful agents can be developed to selectively inhibit the HIV-killing process. Two compounds, the tyrosine kinase inhibitor, herbimycin A, and the phosphatase 2A inhibitor, okadaic acid, were found capable of blocking HIV-mediated killing, although both compounds have substantial toxicity probably limiting any clinical utility. Work continued on developing genetically engineered T cell receptors to direct cytolytic T cells against the HIV-1 envelope proteins. Retroviral vectors encoding chimeric antibody/T cell receptors recognizing either the extracellular or transmembrane HIV envelope glycoprotein were constructed and tested for function. Transgenic mice were produced which were capable of synthesizing chimeric receptor mRNA in their circulating T cell pool.
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