CYTOKINE MEDIATED INHIBITION OF HIV-1 IN LIVER MODEL

为了了解人类免疫缺陷病毒（HIV-1）的潜伏期， 1)感染和各种辅助因子的作用，几个实验 已经开发了模型。 虽然肝源性细胞还没有 它们被认为是HIV-1感染的靶组织，可以作为 重要的模型，因为肝脏异常和高发病率 B型肝炎病毒感染与艾滋病一起发现。 我们观察到， 人肝母细胞瘤HepG2细胞的克隆是CD4阳性的，并且可以被 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， （TNF-α）或佛波醇-12肉豆蔻酸酯-13-乙酸酯（PMA） 在T细胞中复制，这些药物抑制HIV-1复制， 肝细胞 本提案的长期目标和具体目标 1）确定涉及的机制（S） TNF-α对HepG2细胞中HIV-1感染的抑制作用。 比较 TNF-α和PMA对该系统中HIV-1感染影响， 这可能表明HIV-1感染的新途径。2)比较这些 其他肝癌细胞系的数据，包括HepG2克隆， CD4阴性。3)分析HIV-1 DNA和RNA的状态， 被感染的细胞 4)TNF-α诱导基因的分离和鉴定 序列通过消减杂交的HepG2衍生的cDNA文库。 将从PMA处理的HepG2中分离类似的消减cDNA克隆 细胞也是。 这些cDNA将被克隆到真核表达载体中， 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，用于分析其对 HIV-1感染。5)除了毒株III B，我们还将使用HIV-1 前病毒克隆pHXBc2用于感染。 各种稳定细胞系 将分离含有该前病毒基因组的病毒。 TNF-α的作用 α或PMA将在这些感染性克隆中进行评价，这些感染性克隆来源于 各种肝癌细胞系。 蛋白激酶C在肿瘤细胞凋亡中的作用 PMA的影响进行了研究。 6)凝胶阻滞试验和广泛的 DNA足迹法将用于鉴定TNF-α或TNF-α中的蛋白质。 PMA处理的细胞与HIV-1的调节区结合。 7)通过 突变这个前病毒克隆的各个区域，我们打算定位 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，定性，纯化，如果 根据需要，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， 并与处理后的细胞进行比较。 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。

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.