The goal of this proposal is to enhance our understanding of the molecular mechanisms by which cytokines and hormones regulate the production of HIV-1, and the mechanisms which allow certain viral sequences to mediate both positive and negative control of HIV-1 transcription. Initial studies will examine the response of the viral long terminal repeat (LTR) to induction by IL-6, interleukin-6. In order to investigate the nuclear events in the mechanism of HIV-1 activation by IL-6, experiments are proposed to closely evaluate the contributions made by protein binding sites in the LTR to the regulation of HIV- expression by NF-IL6 since this nuclear factor has been implicated in functioning as a key mediator of gene activation by IL-6. Emerging data have provided evidence for a potential synergism between NF-IL6 and NF- kappaB in the mechanisms which control the immune and inflammatory responses. Therefore, experiments are proposed to closely examine the patterns of LTR regulation by IL-1 and IL-6, as well as by appropriate chemical agents. To further identify possible cascade reactions leading to massive activation of HIV-1, experiments will be performed to determine the effects of the potent viral transactivator protein tat on the response of the LTR to multiple cytokines. Evidence suggests that gene activation might be further amplified in response to synergistic effects produced by NF-IL6 and a nuclear receptor. To investigate the contribution of this mechanism to HIV-1 activation, initial studies will evaluate the effects of oestrogen receptors and oestradiol on LTR- mediated transcription, and subsequent experiments will investigate a potential cooperation between NF-IL6 and the oestrogen receptor in the control of HIV-1 expression. A negative regulatory element in the LTR (NRE1) includes a binding site for NF-IL6 overlapping an E-box which provides a binding site for several regulators of gene expression. Current results suggest that NRE1 might correspond to a composite regulator sequence. Studies on selected proteins are proposed in order to understand how this sequence could produce both positive and negative effects on HIV-1 transcription. The experiments in this proposal involve standard procedures in molecular and cell biology. They include mutational analyses, transient expression assays, immunoprecipitation experiments, and DNA binding studies.
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