CYTOKINE MEDIATED INHIBITION OF HIV 1 IN LIVER MODEL

肝脏模型中细胞因子介导的 HIV 1 抑制

• 批准号: 2330808
• 负责人: Ranjit Banerjee
• 金额: $ 8.33万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330808
• 关键词: HTC cell; antiAIDS agent; complementary DNA; cyclic AMP; disease /disorder model; drug screening /evaluation; gel mobility shift assay; gene induction /repression; genetic library; genome; human immunodeficiency virus 1; liver cells; microorganism culture; molecular cloning; nucleic acid repetitive sequence; phorbols; protein kinase C; provirus; subtraction hybridization; transcription factor; transfection /expression vector; tumor necrosis factor alpha; virus DNA; virus RNA; virus genetics

In order to understand the latency of human immunodeficiency virus (HIV-1) infection and the effect of various cofactors, several experimental models have been developed. Although some non-lymphoid cells are not usually considered as the main target tissue for HIV-1 infection, they can serve as important models. Together with other HIV-1 permissive cellular models we have also used human hepatoblastoma HepG2 cells, since hepatic abnormalities and a higher incidence of hepatitis B virus infection are found with AIDS. In contrast to the stimulatory effect of tumor necrosis factor (TNF-alpha) or Phorbol-12-myristate-13-acetate (PMA) in HIV-1 replication in T cells, these agents inhibited HIV-1 replication in liver cells. The long-term objective and specific aims of this proposal are the following: 1) Determine the mechanism(s) involved in inhibition of HIV-1 infection by TNF-alpha in HepG2 cells. Compare the effect of TNF-alpha with that of PMA on HIV-1 infection in this system, which may indicate a novel pathway for HIV-1 infection. 2) Compare these data with other cell lines including the HepG2 clone which is CD4 negative. 3) Analyze the state of the HIV-1 DNA and RNA in these infected cells. 4) Isolate and characterize TNF-alpha and PMA-induced gene sequences by subtraction hybridization of HepG2 derived cDNA libraries. These cDNAs will be cloned in a eukaryotic expression vector so that they can be transfected into various cell lines including HepG2 to obtain stable cell lines for analysis of their resistance to HIV-1 infection. 5) We will use various HIV-1 proviral clones for infection, and stable cell lines containing the proviral genome will be isolated. The effect of TNF-alpha or PMA will be evaluated in these infectious clones derived from various hepatoma cell lines. 6) The role of protein kinase C and cAMP in relation to TNF-alpha and PMA effect will be studied. 7) Gel retardation assays and extensive DNA footprinting will be used to identify the proteins in TNF-alpha or PMA-treated cells which bind to the regulatory regions of HIV-1 and TAR RNA sequences. 8) By mutating various regions of this proviral clone, we intend to localize the region(s) in HIV-1 genome responsible for the TNF- alpha or PMA mediated inhibition. 9) We will identify, characterize, purify, and, if required, isolate the cDNAs encoding the trans-acting proteins from various hepatoma and other cell lines that bind to HIV-1 LTR by screening lambdagt11 library and compare with that of treated cells.

为了了解人类免疫缺陷病毒（HIV-1）的潜伏期， 感染和各种辅助因子的作用，几种实验模型 已经被开发出来了。 虽然一些非淋巴细胞通常不 被认为是HIV-1感染的主要靶组织， 作为重要的模特。 与其他HIV-1容许细胞模型一起 我们还使用了人肝母细胞瘤HepG 2细胞，因为肝母细胞瘤HepG 2细胞是由人肝母细胞瘤HepG 2细胞引起的。 乙型肝炎病毒（B）感染的发病率较高。 发现艾滋病。 与肿瘤坏死的刺激作用相反 因子（TNF-α）或佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA） 这些药物抑制HIV-1在肝脏中的复制， 细胞 这项建议的长远目标和具体目标是 1）确定涉及抑制HIV-1的机制 HepG 2细胞中TNF-α的感染。 比较TNF-α的作用 PMA对HIV-1感染的抑制作用可能与PMA对HIV-1感染的抑制作用有关。 HIV-1感染的新途径。 2)将这些数据与其他单元格进行比较 包括CD 4阴性的HepG 2克隆的细胞系。 3)分析 这些感染细胞中HIV-1 DNA和RNA的状态。 4)分离和 通过减法表征TNF-α和PMA诱导的基因序列 HepG 2衍生的cDNA文库的杂交。 这些cDNA将被克隆 在真核表达载体中，以便它们可以被转染到 包括HepG 2在内的各种细胞系，以获得稳定的细胞系， 分析其对HIV-1感染的抵抗力。 5)我们将使用各种 用于感染的HIV-1前病毒克隆，和含有所述前病毒克隆的稳定细胞系。 分离前病毒基因组。 TNF-α或PMA的作用将是 在这些来自各种肝癌细胞的感染性克隆中进行了评价 线 6)蛋白激酶C和cAMP与TNF-α的关系 并研究PMA效应。 7)凝胶阻滞试验和广泛的 DNA足迹法将用于识别TNF-α或TNF-α中的蛋白质 结合HIV-1和TAR调节区的PMA处理细胞 RNA序列。 8)通过突变这个前病毒克隆的各个区域， 旨在定位HIV-1基因组中负责TNF-α的区域， α或PMA介导的抑制。 9)我们将识别，描述， 纯化，并且如果需要，分离编码反式作用的cDNA。 与HIV-1 LTR结合的来自各种肝癌和其他细胞系的蛋白质 通过筛选cDNA文库并与处理细胞比较。