The goal of this research is to understand common steps in the pathways of cytokine signalling for HIV proviral activation and virus replication in blood cells and to develop effective inhibitory strategies.
The specific aims are: (1) To elucidate pathways involved in cytokine signalling for HIV activation in hematopoietic cells. (2) To develop strategies for inhibiting cytokine-mediated HIV activation and preventing release from latency. (3) To test gene transfer strategies to inhibit viral replication in myelomonocytic cells. Pathways of cytokine signalling for HIV proviral activation and virus production are studied in model cell lines representing the T lymphocyte (ACH-2), the mononuclear phagocyte (U-l), and the myeloid progenitor (J22HL-60). The roles of the neutral and acid sphingomyelinase signalling pathways will be evaluated, separating effects of TNF produced in an autocrine fashion using antibody to TNF with blocking antibodies to the 55- and 75-kD TNF receptors. The HIV signalling pathways of GM-CSF, IL-1, IL-3, IL-4, IL-6, M-CSF, G-CSF, and endotoxin will be examined in a manner isolating direct effects from those due to autocrine TNF production. Results observed in the cell line models will be extended to normal cell counterparts (T lymphocytes, and myeloid precursors) assessing infectibility and transcription using measures of viral production, LTR reporter gene activity, and RT-PCR transcript analysis. A cytokine signalling proviral activation model system will be established in Xenopus laevis oocytes using an intranuclear HIV LTR luciferase construct with reconstitution of a TNF signalling pathway by cytoplasmic translation of RNA for the TNF receptors. Several classes of TNF inhibitors will be examined in the cell line and Xenopus systems, as well as potential receptor antagonists and lipid pathway inhibitors. Redox state regulation of HIV transcription will be evaluated in the cell lines and Xenopus oocytes using a vitamin C transport system facilitated by the glucose transporters as well as other redox regulators such as vitamin E and phenolic lipid anti-oxidants. Methods for directly inhibiting proviral transcription and viral replication will be approached in mononuclear phagocytes with gene transfer studies using retroviral constructs expressing TAR sequences and the rev decoy, RRE. The gene therapy models will examine both primary infectibility and release from latency. These investigations aim to establish a scientific basis for biologic interventions capable of inhibiting HIV replication which can translate into means for maintaining the asymptomatic state in HIV- infected persons.
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