This project will investigate regulation of chronic human immunodeficiency virus-1 (HIV-1) infection in the human monocyte and CD4+ T lymphocyte. We will concentrate on agents which block induction of virus in these cells as potential clinical therapeutics. We will focus on two classes of inhibitory molecules: 1) gonadal steroids and their antagonists which share protein kinase C inactivator and potential LTR-enhancer binding properties and 2) glucocorticoids capable of interacting with adrenal steroid-responsive elements in the HIV-LTR and. In preliminary experiments we utilized models for regulation of HIV-1 infection in T lymphoblastoid and monocyte lines, delineating their relationship to protein kinase activation and tat-mediated trans-activation. We found that PKC-specific inhibitors abrogated phorbol ester-mediated upregulation of HIV-1 replication. In addition, as adrenal and gonadal steroids can alter specific gene expression in another immunosuppressive retrovirus, mouse mammary tumor virus, we explored the ability of such hormones to regulate HIV replication and modify transcription at the HIV-1 LTR. We found that two such agents could block the phorbol ester-mediated upregulation of HIV, concomitantly suppressing transcriptional enhancement of an HIV-LTR linked reporter gene, while leaving basal levels of HIV-LTR directed transcription unperturbed. Potential relationships to consensus sequences for hormone responsive elements (HRE) in the HIV-LTR were noted. One particularly effective gonadal steroid antagonist had both steroid binding potential and the capacity to inhibit PKC. In the latter phase of this project, indications of potential in vivo efficacy of such inhibitors will be sought from a study of their interactions with viral inducers administered to transgenic mice containing HIV-LTR-CAT and HIV-tat interactions in vivo. These animals have been made available to us through a collaboration with Dr. Heiner Westphal at the NIH. Exploration of the mechanism of action of these agents, employing techniques already familiar to the investigators, such as trans-activation assays, radioimmunoprecipitation of phosphorylated proteins, deletion mutagenesis, DNA sequencing, use of modified, """"""""anti-sense"""""""" oligomers, and gel retardation, should lead to the rational design of agents capable of suppressing activation of chronic HIV infection.
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