Transcriptional regulation of HIV-1 involves a complex interplay between exogenous signals, viral and host cell proteins, and regulatory elements in the long terminal repeat (LTR). The primary objective of this proposal is to identify physiologic signals that may modulate HIV-1 gene expression in T lymphocytes and monocytes. Towards this goal, it was discovered that five steroid hormone receptors with currently unidentified ligands, ARP-1, EAR-2, EAR-3, HNF-4, and NGFI-B, can bind to a negative regulatory element (NRE) in the HIV-1 LTR. These findings suggest that multiple signal transduction pathways may converge onto the HIV-1 NRE and raise the intriguing possibility that HIV-1 gene expression may be modulated by currently unidentified hormones or ligands. In addition, RXRalpha (9-cis retinoic acid receptor alpha) forms heterodimers with ARP-1, EAR-2, EAR-3, and RARalpha (all-trans retinoic acid receptor alpha) which bind to the HIV-1 NRE, suggesting that HIV-1 gene expression may be modulated by 9-cis and all-trans retinoic acid. Transient transfection experiments showed that ARP-1 and EAR-3 downregulated HIV-1 expression in HeLa cells. However, the effects of the receptors that bind to the NRE on HIV expression in T cells and monocytes are not known.
The specific aims are: 1) To determine the effects of ARP-1, EAR-2, EAR- 3, HNF-4, RXRalpha, RARalpha, and NGFI-B overexpression on HIV-1 LTR- driven transcription in T lymphocytes and monocytes. 2) To determine whether the HIV-1 NRE is a functional steroid responsive element. 3) To determine the effects of certain cytokines and growth factors on the expression of the steroid receptors that bind to the NRE in T cells and monocytes. The experimental design and methods for achieving these goals are: 1) Cotransfections of plasmids expressing the above receptors with constructs containing the CAT reporter gene under the control of the HIV- 1 LTR in T cells (Jurkat and MOLT-4), and monocytic cells (U937 and THP- 1), using the electroporation and DEAE-dextran methods. Transfections with RXRalpha and RARalpha will include post-transfection treatment of the cells with 9-cis and all-trans retinoic acid. The CAT activity will be used to evaluate the effects of the receptors on the HIV-1 LTR-driven transcription. 2) cotransfections of the above receptors with constructs containing the CAT gene under the control of the HIV-1 NRE cloned upstream of a heterologous promoter will determine the potential of the NRE to respond to steroid receptors in vivo. 3) Northern blot analysis will determine the expression pattern of these receptors in T cells and monocytes before and after treatment with TNF-alpha, GM-CSF, M-CSF, PDGF, and NGF. The studies proposed here may reveal novel signal transduction pathways that contribute to HIV-1 latency versus productive infection. In addition, this information may provide a basis upon which new therapeutic agents that inhibit HIV-1 replication could be developed.
|Ladias, J A (1994) Convergence of multiple nuclear receptor signaling pathways onto the long terminal repeat of human immunodeficiency virus-1. J Biol Chem 269:5944-51|