NF-KB/REL AS AN ACTIVATOR OR INHIBITOR OF HIV-1 GROWTH

初次感染1型人类免疫缺陷病毒(HIV-1)， 获得性免疫缺陷综合征(AIDS)的病原体 通常之后会有一段较长的无症状期 显性疾病的出现。HIV-1全程复制动态 这一临床连续体似乎严重依赖于 通常诱导的特定宿主反式调节因子 对免疫刺激的反应。除了对其高度亲和力 逆转录病毒5‘末端存在功能性增强子序列 重复(LTR)，核因子-kappaB转录因子被快速诱导 在T细胞激活过程中，可能代表着一种主要的宿主调节 调控人类免疫缺陷病毒-1基因表达的途径 持续感染的CD4+T淋巴细胞。分子克隆研究已经 核因子-kappaB的50kD(P50)和65kD(P65)亚基均为 与v-rel癌蛋白具有惊人的N-末端序列同源性， 它的正常细胞对应物(c-rel)和腹侧形态原背侧 在果蝇身上。特别是，p65是一种有效的转录激活因子。 也是IkappaB的“受体”，a 核因子-kappaB功能的细胞质抑制物。与之形成鲜明对比的是，C-Rel 作为HIV-1 LTR定向转录的抑制物，因此可能 与促进病毒的一种反调节宿主细胞因子相对应 衰减。这些拟议研究的主要目标是探索 生理调节、作用机制和生物学作用 这些不同的Rel多肽对HIV-1基因的调控作用 表情。为了实现这一总体目标，一个全面的 将进行突变分析，以确定并彻底剖析 P65中存在的功能域，它们是 反式激活，HIV-1增强子结合，IkappaB介导的抑制，以及 多聚合化。反过来，选定的表型变体将用于 寻找新的核因子-kappaB信号的“相互克隆”策略 参与介导HIV-1转录激活的成分或 通过蛋白质/蛋白质相互作用进行抑制。探索核因子的作用- KappaB在控制HIV-1转录调控机制中的作用 在体内表达，将使用单特异性抗肽抗体 为了从生物化学的角度定义存在于 组成主要靶点的原代CD4+表达细胞 HIV-1感染。结合体外转录和体内转录 足迹分析，这些抗体也将被用来定义 HIV-1在已建立细胞中表达减弱的生化基础 病毒潜伏期的培养模型。最后，稳定转染人T细胞 将产生有条件地表达p65或c- 通过与多种类固醇的配体结合域融合来释放 激素受体，以便直接确定它们的相对 HIV-1感染后的转录效应。加在一起，这些 拟议的实验方法应产生基本的机械论 对核因子-kappaB/Rel转录的两个组成部分的洞察 因子家族及其在调节中的潜在不同作用 HIV-1基因的表达。因此，这些信息可能是 了解核因子-kappaB在转录中的精确参与 持续和有效地控制HIV-1复制的计划 被感染的细胞。

Primary infection with the type 1 human immunodeficiency virus (HIV-1), the etiologic agent of the Acquired Immune Deficiency Syndrome (AIDS), is typically followed by an extended asymptomatic period before the emergence of overt disease. the replication dynamics of HIV-1 throughout this clinical continuum appear critically dependent on the action of specific host trans-regulatory factors that are normally induced in response to immunologic stimuli. In addition to its high affinity for functional enhancer sequences present in the retroviral 5' long terminal repeat (LTR), the NF-kappaB transcription factor is rapidly induced during T cell activation and likely represents a major host regulatory pathway governing the onset of productive HIV-1 expression in persistently infected CD4+ T lymphocytes. Molecular cloning studies have revealed that the 50 kD (p50) and 65 kD (p65) subunits of NF-kappaB both share striking N-terminal sequence homology with the v-Rel oncoprotein, its normal cellular counterpart (c-Rel), and dorsal, a ventral morphogen in Drosophila. In particular, p65 is a potent transcriptional activator of the HIV-1 LTR and also serves as a "receptor" for IkappaB, a cytoplasmic inhibitor of NF-kappaB function. In sharp contrast, c-Rel acts as a repressor of HIV-1 LTR-directed transcription and may thus correspond to a counter-regulatory host cell factor that promotes viral attenuation. the principal goal of these proposed studies is to explore the physiological regulation, mechanism of action, and biological role of these various Rel polypeptides in the modulation of HIV-1 gene expression. To approach this overall objective, a comprehensive mutational analysis will be undertaken to define and completely dissect the functional domains present in p65 that are required for transactivation, HIV-1 enhancer binding, IkappaB-mediated inhibition, and multimerization. In turn, selected phenotypic variants will be used in an "interaction cloning" strategy to identify novel NF-kappaB signaling components involved in mediating HIV-1 transcriptional activation or repression via protein/protein interactions. to explore the role of NF- kappaB in transcriptional regulatory mechanisms controlling HIV-1 expression in vivo, monospecific anti-peptide antibodies will be employed to biochemically define the spectrum of Rel polypeptides present in primary CD4+ expressing cells that comprise the principal targets for HIV-1 infection. In conjunction with in vitro transcription and in vivo footprinting analyses, these antibodies will also be used to define the biochemical basis for attenuated HIV-1 expression in established cell culture models of viral latency. Finally, stably transfected human T cell lines will be generated that conditionally express either p65 or c- Rel by fusion to the ligand binding domains of a variety of steroid hormone receptors in order to directly determine their relative transcriptional effects following HIV-1 infection. Together, these proposed experimental approaches should yield fundamental mechanistic insights into two integral components of the NF-kappaB/Rel transcription factor family an their potentially divergent roles in the regulation of HIV-1 gene expression. As such, this information may be key to understanding the precise involvement of NF-kappaB in the transcriptional programs controlling HIV-1 replication in persistently and productively infected cells.