HIV AND NF-KB INTERACTIONS IN MONOCYTES

人类巨噬细胞是艾滋病毒持续复制的主要储存库。 然而，调节病毒在这些细胞中持续存在的机制 未知。他们的特征可以导致根除这一现象 关键的病毒库。核因子kB是一种转录因子 它调控着HIV的转录。在一个持续性艾滋病毒的模型中 感染单核细胞和巨噬细胞，但不感染T淋巴细胞，我们有 证明了核因子-kB是在脑出血过程中被特异性诱导的。 感染。这种细胞特异性病毒相互作用对 免疫功能和关于艾滋病毒复制的问题仍不清楚。此外， 细胞外刺激或HIV持续复制的机制 对单核细胞中的核因子-kB的调节作用尚不清楚。我们的研究是 旨在刻画HIV中调节核因子-kB的机制- 感染的单核细胞和巨噬细胞。这些研究的结果将 使我们能够确定核因子-kB在病毒持续中的相对作用。 提供了初步数据，重点是转录和后转录 转录调控核因子-kB的机制。第一，核因子-kB的增加 HIV感染的单核细胞的活性仅次于合成增加 P105/p50核因子-kB亚基的表达。我们克隆的基因启动子 编码p105表明其受核因子-kB的自动调节。因此，艾滋病毒和核因子-kB 通过其p50亚基参与人类体内的双重自我调节环路 单核细胞。对该启动子调控区域的详细研究 HIV和细胞外刺激通过这种途径增加其转录 单核细胞的活性将被执行。第二，增加核武器 感染艾滋病毒的单核细胞中的易位遵循特定的丝氨酸- 核因子-kB抑制因子MAD3的苏氨酸磷酸化。一种新的信号 在HIV中上调的具有功能的单核细胞信号转导通路- 感染细胞，磷脂酰胆碱-磷脂酶C(PC-PLC)，结果 在核因子-kB的激活中。这条途径中的一个激酶，PKC-Zeta((Zeta)是 HIV介导的NF-kB激活和磷酸化MAD3所必需的 体外培养。此外，已知与PC-PLC相连的ras-RAF通路 PKC-Zeta还介导HIV依赖的核因子-kB的激活。一个系统的 分析这些通路的生化步骤，包括激酶 这一磷酸化的IKB将被执行。在感染艾滋病毒的单核细胞中 巨噬细胞，这些假定的信号转导途径导致 MAD3在体内的丝氨酸-苏氨酸磷酸化及其作用 随后的蛋白质分解。这增加了核因子-kB的核转位， 不仅激活p105/p50和HIV-LTR的转录，而且还 MAD3。因此，HIV、NF-kB(p150/p50)和MAD3现在形成了一个连续的 人单核巨噬细胞的三重自我调节环。抑制 以上确定的具体机制应确认和定义其 在调节核因子-kB和单核细胞病毒复制中的作用 巨噬细胞。这将尝试使用互补性和替代性 接近了。 通过这种多学科的方法，我们希望确定 未感染和HIV感染患者中调节核因子-kB的分子机制 单核细胞和巨噬细胞，以便更好地了解核因子-2的作用。 KB在病毒的持久性、潜伏期和免疫功能方面 细胞。

Human macrophages are a major reservoir of persistent HIV replication. However, the mechanisms that regulate viral persistence in these cells ar unknown. Their characterization could lead to the eradication of this key viral reservoir. Nuclear factor kB (NF-kB) is a transcription factor that regulates the transcription of HIV. In a model of persistent HIV infection in monocytes and macrophages but not in T lymphocytes, we have demonstrated that NF-kB is specifically induced during the course of the infection. The impact of this cell specific-virus interaction on the immune function and on HIV replication remains unknown. Furthermore, the mechanisms by which extracellular stimuli or persistent HIV replication regulate NF-kB in monocytes are poorly characterized. Our research is directed at characterizing the mechanisms that regulate NF-kB in HIV- infected monocytes and macrophages. Results from these studies will enable us to determine the relative role of NF-kB in viral persistence. Preliminary data is provided focusing on transcriptional and post- transcriptional mechanisms regulating NF-kB. First, increased NF-kB activity in HIV-infected monocytes is secondary to increased synthesis of the p105/p50 NF-kB subunit. Our cloning of the promoter of the gene encoding p105 indicates its autoregulation by NF-kB. Thus, HIV and NF-kB through its p50 subunit engage in a double autoregulatory loop in human monocytes. Detailed studies of the regulatory regions of this promoter through which HIV and extracellular stimuli increase its transcriptional activity in monocytes will be performed. Second, increased nuclear translocations in HIV-infected monocytes follows a specific serine- threonine phosphorylation of the NF-kB inhibitor, MAD3. A novel signal transduction pathway that is functional monocytes and upregulated in HIV- infected cells, phosphatidyl choline-phospholipase C (PC-PLC), results in NF-kB activation. A kinase within this pathway, PKC-zeta ((zeta) is required for HIV-mediated NF-kB activation and phosphorylates MAD3 in vitro. Moreover, the ras-raf pathway, known to be connected to PC-PLC and PKC-zeta also mediates HIV-dependent NF-kB activation. A systematic analysis of the biochemical steps of these pathways, including kinases that phosphorylate IkB will be performed. In HIV-infected monocytes and macrophages, these putative signal transduction pathways result in a specific serine-threonine phosphorylation of MAD3 in vivo with its subsequent proteolysis. This increases NF-kB nuclear translocation which not only activates transcription of p105/p50 and the HIV-LTR, but also of MAD3. Thus, HIV,NF-kB (p150/p50) and MAD3 now form a continuous triple autoregulatory loop in the human monocyte-macrophage. Inhibition of specific mechanisms identified above should confirm and define their role in the regulation of NF-kB and viral replication in monocytes- macrophages. This will be attempted using complementary and alternative approaches. Through this multidisciplinary approach, we expect to identify the molecular mechanisms that regulate NF-kB in uninfected and HIV-infected monocytes and macrophages, in order to better understand the role of NF- kB in viral persistence, latency and in the immune function of these cells.