REGULATION OF HIV-1 GENE EXPRESSION BY STEROID RECEPTORS

HIV-1的转录调控涉及一种复杂的相互作用 外源信号、病毒和宿主细胞蛋白以及调节元件 在长末端重复序列(LTR)。这样做的主要目标是 建议识别可能调节HIV-1基因的生理信号 在T淋巴细胞和单核细胞中表达。为了这个目标，它是 发现五种类固醇激素受体目前 ARP-1、EAR-2、EAR-3、HNF-4和NGFI-B等未知配体可以结合 与HIV-1 LTR中的负调控元件(NRE)有关。这些发现 提示多条信号转导通路可能汇聚到 HIV-1 NRE和提高了HIV-1基因表达的耐人寻味的可能性 可能受目前未知的激素或配体的调节。在……里面 此外，RXRpha(9-顺式维甲酸受体α)形成 含ARP-1、EAR-2、EAR-3和RARAlpha(全反式维甲酸)的杂二聚体 与HIV-1 NRE结合的酸性受体α)，表明HIV-1 基因表达可能受9-顺式和全反式维甲酸的调节。 瞬时转染实验显示ARP-1和EAR-3 下调HeLa细胞中HIV-1的表达。然而，它的影响 与NRE结合的受体对T细胞和HIV表达的影响 单核细胞是未知的。 具体目标是：1)确定ARP-1、EAR-2、EAR-1的作用。 3、HNF-4、RXRpha、RARpha和NGFI-B在HIV-1 LTR上的过表达- 驱动T淋巴细胞和单核细胞转录。2)确定 HIV-1NRE是否是功能性类固醇反应元件。3)至 确定某些细胞因子和生长因子对 与NRE结合的类固醇受体在T细胞和 单核细胞。 实现这些目标的实验设计和方法如下：1) 表达上述受体的真核表达载体与 含有HIV控制下的猫报告基因的构建- 1 T细胞(Jurkat和MOLT-4)和单核细胞(U937和THP-4)的LTR。 1)、电穿孔法和DEAE-葡聚糖法。传播学 RXRpha和RARpha将包括转基因后治疗 含9-顺式和全反式维甲酸的细胞。CAT活动将 用来评价受体在HIV-1 LTR驱动中的作用 抄写。2)将上述受体与真核表达载体共转染 克隆的HIV-1 NRE控制下的CAT基因 异源启动子的上游将决定 NRE对体内的类固醇受体有反应。3)Northern杂交分析 将决定这些受体在T细胞中的表达模式 单核细胞经肿瘤坏死因子-α、粒-巨噬细胞集落刺激因子、巨噬细胞集落刺激因子、血小板衍生生长因子治疗前后 和NGF。 本文提出的研究可能揭示新的信号转导途径。 与生产性感染相比，这会导致HIV-1潜伏期。在……里面 此外，这一信息可以提供新的治疗方法的基础 可以开发抑制HIV-1复制的试剂。

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.