REGULATION OF HIV-1 GENE EXPRESSION BY STEROID RECEPTORS

HIV-1的转录调控涉及以下因素之间复杂的相互作用： 外源信号、病毒和宿主细胞蛋白以及调节元件 长末端重复序列（LTR） 本研究的主要目的 一项旨在鉴定可能调节HIV-1基因的生理信号的研究 在T淋巴细胞和单核细胞中表达。 为了实现这一目标， 发现五种类固醇激素受体， 未鉴定的配体阿普-1、HNF-2、HNF-3、HNF-4和NGFI-B可以结合 与HIV-1 LTR中的负调控元件（NRE）相关联。 这些发现 表明多种信号转导途径可能会汇聚到 HIV-1 NRE，并提出了有趣的可能性，HIV-1基因表达 可能是由目前未鉴定的激素或配体调节的。 在 此外，RXR α（9-顺式视黄酸受体α）形式 具有阿普-1、RAR-2、RAR-3和RAR α的异二聚体（全反式视黄酸 酸受体α），其结合HIV-1 NRE，表明HIV-1 基因表达可由9-顺式和全反式视黄酸调节。 瞬时转染实验表明，阿普-1和ARP-3 下调HeLa细胞中HIV-1的表达。 然而， 在T细胞中HIV表达上与NRE结合的受体， 单核细胞未知。 具体目的是：1）确定阿普-1、ARP-2、ARP-3、ARP-4、ARP-5、ARP-6、ARP-7、ARP-8、ARP-9、ARP-10、ARP-11、ARP-12、ARP-13、ARP-14、ARP-15、ARP-16、ARP-17、ARP-18、ARP-19、ARP- HIV-1 LTR上HNF-3、HNF-4、RXR α、RAR α和NGFI-B过表达- 在T淋巴细胞和单核细胞中驱动转录。 2)以确定 HIV-1 NRE是否是功能性类固醇反应元件。 3)到 确定某些细胞因子和生长因子对 在T细胞中结合NRE的类固醇受体的表达， 单核细胞 实现这些目标的实验设计和方法是：1） 表达上述受体的质粒与 含有在HIV-1控制下的CAT报告基因的构建体， 在T细胞（Jurkat和MOLT-4）和单核细胞（U937和THP-1）中的LTR 1），采用电穿孔法和DEAE-dextran法。 转染 RXR α和RAR α的转染后治疗将包括 用9-顺式和全反式维甲酸处理细胞。 CAT活动将 用于评估受体对HIV-1 LTR驱动的 转录。 2)上述受体与构建体的共转染 含有受HIV-1 NRE控制的CAT基因， 异源启动子的上游将决定异源启动子的潜力。 NRE对体内类固醇受体有反应。 3)北方印迹分析 将决定这些受体在T细胞中的表达模式， 在用TNF-α、GM-CSF、M-CSF、PDGF治疗前后的单核细胞， 和NGF。 本文提出的研究可能揭示新的信号转导途径 导致HIV-1潜伏感染与生产性感染的差异。 在 此外，这些信息可以提供新的治疗方法的基础。 可以开发抑制HIV-1复制的药物。

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.