CYTOKINE MEDIATED INHIBITION OF HIV-1 IN LIVER MODEL

肝模型中细胞因子介导的 HIV-1 抑制

• 批准号: 3460462
• 负责人: Ranjit Banerjee
• 金额: $ 12.26万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 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 1994-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3460462
• 关键词: DNA binding protein; DNA footprinting; HIV infections; RNA binding protein; clone cells; complementary DNA; gel electrophoresis; hepatoblastoma; human immunodeficiency virus 1; latent virus infection; liver infection; molecular cloning; neoplastic cell culture for noncancer research; nucleic acid sequence; phorbols; protein kinase C; provirus; site directed mutagenesis; subtraction hybridization; transcription factor; transfection; tumor necrosis factor alpha; virus DNA; virus RNA; virus replication

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 cells of liver origin are not yet considered as target tissue for HIV-1 infection, they can serve as an important model, since hepatic abnormalities and a high incidence of hepatitis B virus infection are found with AIDS. We observed that a clone of human hepatoblastoma HepG2 cells are CD4 positive and can be infected with HIV-1 and support HIV-1 replication by producing infection virions. 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 mechanisms(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 hepatoma 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 induced gene sequences by subtraction hybridization of HepG2 derived cDNA libraries. Similarly subtracted cDNA clones will be isolated from PMA treated HepG2 cells as well. These cDNAs will be cloned in an 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) In addition to the strain III B, we will use HIV-1 proviral clone pHXBc2 for infection. Various stable cell lines containing this 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. The role of protein kinase C in relation to PMA effect will be studied. 6) Gel retardation assay 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. 7) 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. 8) We will identify, characterize, purify, and if required, isolate the cDNAs encoding the trans-acting proteins from various hepatoma cell lines that bind to HIV-1 LTR by screening a lambdagt11 library and compare with that of treated cells. 9) The binding of TNF-alpha and PMA treated HepG2 nuclear protein to HIV-1 TAR RNA sequences will be studied.

为了了解人类免疫缺陷病毒（HIV- 1）感染和各种辅因子的影响，几个实验 模型已经开发出来。 尽管肝脏来源的细胞尚未 被认为是 HIV-1 感染的靶组织，它们可​​以作为 重要的模型，因为肝脏异常和高发病率 乙型肝炎病毒感染被发现患有艾滋病。 我们观察到一个 人肝母细胞瘤 HepG2 细胞的克隆呈 CD4 阳性，可 感染 HIV-1 并通过产生感染来支持 HIV-1 复制 病毒体。 与肿瘤坏死因子的刺激作用相反 HIV-1 中的 (TNF-α) 或佛波醇 12 肉豆蔻酸 13 乙酸酯 (PMA) T 细胞中的复制，这些药物抑制了 HIV-1 在 T 细胞中的复制 肝细胞。 本提案的长期目标和具体目标 如下： 1) 确定涉及的机制 HepG2 细胞中 TNF-α 抑制 HIV-1 感染。 比较 TNF-α 与 PMA 对该系统中 HIV-1 感染的影响， 这可能表明 HIV-1 感染的新途径。 2）比较这些 与其他肝癌细胞系（包括 HepG2 克隆）的数据 CD4阴性。 3) 分析这些细胞中 HIV-1 DNA 和 RNA 的状态 被感染的细胞。 4) TNF-α诱导基因的分离和表征 通过 HepG2 衍生的 cDNA 文库的消减杂交获得序列。 将从 PMA 处理的 HepG2 中分离出类似的消减 cDNA 克隆 细胞也是如此。 这些 cDNA 将被克隆到真核表达中 载体，以便它们可以转染到各种细胞系中，包括 HepG2获得稳定的细胞系以分析其耐药性 HIV-1 感染。 5) 除了 III B 株外，我们还将使用 HIV-1 用于感染的前病毒克隆 pHXBc2。 各种稳定细胞系 含有该原病毒基因组的病毒将被分离出来。 TNF-α的作用 α 或 PMA 将在这些源自以下来源的感染性克隆中进行评估 各种肝癌细胞系。 蛋白激酶C的作用与 对 PMA 的影响将进行研究。 6) 凝胶阻滞测定和广泛 DNA 足迹法将用于识别 TNF-α 或 PMA 处理的细胞与 HIV-1 的调节区域结合。 7) 通过 突变这个原病毒克隆的各个区域，我们打算本地化 HIV-1 基因组中负责 TNF-α 或 PMA 的区域 介导的抑制。 8) 我们将鉴定、表征、纯化，并且如果 需要时，从其中分离编码反式作用蛋白的 cDNA 通过筛选与 HIV-1 LTR 结合的各种肝癌细胞系 lambdagt11 文库并与处理过的细胞进行比较。 9) 的 TNF-α 和 PMA 处理的 HepG2 核蛋白与 HIV-1 TAR 的结合 将研究RNA序列。