HIV AND NF-KB INTERACTIONS IN MONOCYTES

人类巨噬细胞是HIV持续复制的主要储存库。 然而，调节这些细胞中病毒持久性的机制 未知。 他们的特点可能会导致根除这种 关键的病毒库 核因子kB（NF-κ B）是一种转录因子 调节艾滋病病毒转录的基因 在一个艾滋病毒持续存在的模型中， 感染单核细胞和巨噬细胞，但不是在T淋巴细胞，我们有 证明了NF-kB是特异性诱导的过程中， 感染 这种细胞特异性-病毒相互作用对细胞增殖的影响， 免疫功能和艾滋病毒复制仍然未知。 而且 细胞外刺激或持续的HIV复制 调节单核细胞中的NF-kB的特征很差。 我们的研究是 旨在表征调节HIV中NF-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NF-κ B在病毒持久性中的相对作用。 提供了初步数据，重点是转录和后， 转录机制调节NF-κ B。 第一，NF-kB增加 HIV感染的单核细胞中的活性是继发于合成增加 p105/p50 NF-kB亚基的表达。 我们克隆的基因启动子 编码p105表明其通过NF-κ B的自身调节。 因此，HIV和NF-kB 通过其p50亚单位参与人的双自动调节环， 单核细胞 对该启动子调控区的详细研究 通过HIV和细胞外刺激增加其转录 将进行单核细胞中的活性。 第二，增加核 HIV感染的单核细胞中的易位遵循特定的丝氨酸- NF-kB抑制剂MAD3的苏氨酸磷酸化。 一个新的信号 转导途径，是功能性单核细胞，并在HIV-1中上调。 感染细胞，磷脂酰胆碱-磷脂酶C（PC-PLC），结果 在NF-κ B活化中。 该途径中的激酶PKC-zeta（（zeta）是 HIV介导的NF-kB激活所需的蛋白质，并磷酸化MAD3， 体外 此外，已知与PC-PLC相关的ras-raf途径 PKC-zeta也介导HIV依赖的NF-κ B活化。 一个系统 分析这些途径的生化步骤，包括激酶 将进行IkB磷酸化。 在HIV感染的单核细胞中， 巨噬细胞，这些假定的信号转导途径导致 MAD3体内特异性丝氨酸-苏氨酸磷酸化及其 随后的蛋白水解。 这增加了NF-kB核转位， 不仅激活p105/p50和HIV-LTR的转录，而且 在MAD3 因此，HIV、NF-kB（p150/p50）和MAD 3现在形成了一个连续的 人单核细胞-巨噬细胞的三重自动调节环。 抑制 应确认和界定其 在单核细胞中调节NF-κ B和病毒复制中的作用- 巨噬细胞 这将尝试使用补充和替代 接近。 通过这种多学科的方法，我们希望确定 在未感染和HIV感染者中调节NF-kB的分子机制 单核细胞和巨噬细胞，为了更好地了解NF-κ B的作用， kB在病毒的持久性，潜伏期和免疫功能，这些 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.