CYTOKINASE MEDIATED INHIBITION OF HIV-1 IN LIVER MODEL

为了了解人类免疫缺陷病毒(HIV)的潜伏期。 1)感染和各种辅助因素的影响，几个实验 模型已经被开发出来。尽管肝脏来源的细胞还没有 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的 感染了乙肝病毒的人被发现患有艾滋病。我们观察到一个 人肝母细胞瘤细胞系HepG2的克隆是CD4阳性的，可以 感染HIV-1并通过产生感染来支持HIV-1复制 病毒粒子。与肿瘤坏死因子的刺激作用相反 HIV-1中的肿瘤坏死因子-α或佛波醇-12-肉豆蔻酸酯-13-醋酸酯(PMA) 在T细胞中复制，这些药物抑制HIV-1在 肝细胞。这项建议的长期目标和具体目标 主要内容如下：1)确定(S)参与的机制 肿瘤坏死因子-α对人肝癌细胞HIV-1感染的抑制作用比较一下 肿瘤坏死因子-α联合PMA对HIV-1感染的影响 这可能为HIV-1感染提供了一条新的途径。2)比较这些 与其他肝癌细胞系的数据，包括HepG2克隆 CD_4阴性。3)分析这些病毒中HIV-1DNA和RNA的状态 被感染的细胞。4)分离和鉴定肿瘤坏死因子-α诱导基因 利用消减杂交技术对HepG2衍生的cDNA文库进行测序。 将从PMA处理的HepG2中分离出类似的消减后的cDNA克隆 细胞也是如此。这些cDNA将被克隆到真核表达中 载体，以便它们可以被转染到各种细胞系中，包括 HepG2获得稳定的细胞系用于分析其抗药性 HIV-1感染。5)除了III B毒株外，我们还将使用HIV-1 用于感染的前病毒克隆PHXBc2。各种稳定的细胞系 包含这一前病毒基因组的病毒将被分离。肿瘤坏死因子的作用- 阿尔法或PMA将在这些来自 各种肝癌细胞系。蛋白激酶C在细胞周期调控中的作用 对PMA的影响进行了研究。6)凝胶阻滞法和广泛性 DNA足迹将用于识别肿瘤坏死因子-α或 PMA处理的细胞与HIV-1的调节区结合。7)由 突变这个前病毒克隆的不同区域，我们打算本地化 HIV-1基因组中负责肿瘤坏死因子-α或PMA的区域(S) 中介抑制。8)我们将识别、表征、净化，如果 必需的，分离编码反式作用蛋白的cDNA 通过筛选与HIV-1 LTR结合的多种肝癌细胞系 Lambdagt11文库，并与处理细胞进行比较。9) 肿瘤坏死因子-α和PMA处理的HepG2核蛋白与HIV-1 TAR的结合 将对RNA序列进行研究。

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.