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 感染及多种辅因子的影响，几种实验模型 已经被开发出来了。尽管一些非淋巴样细胞通常不是 被认为是HIV-1感染的主要目标组织，它们可以 作为重要的典范。与其他HIV-1允许的细胞模型一起 我们还使用了人肝母细胞瘤HepG2细胞，因为肝细胞 乙肝病毒感染的异常和较高的发病率 被发现患有艾滋病。与肿瘤坏死的刺激作用相反 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 在T细胞中复制，这些药物抑制HIV-1在肝脏中的复制 细胞。这项建议的长期目标和具体目标是 1)确定抑制HIV-1病毒的机制(S) 肿瘤坏死因子-α在HepG2细胞中的感染。比较肿瘤坏死因子-α的作用 在这个系统中，PMA对HIV-1感染的影响可能表明 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CD4阴性的HepG2克隆系。3)分析 这些感染细胞中HIV-1DNA和RNA的状态。4)隔离和 用差减技术鉴定肿瘤坏死因子-α和PMA诱导的基因序列 HepG2衍生的cDNA文库的杂交。这些cDNA将被克隆 在真核表达载体中，这样它们就可以被转染到 包括HepG2在内的各种细胞系获得稳定的细胞系 他们对HIV-1感染的抵抗力分析。5)我们将使用各种 用于感染的HIV-1前病毒克隆和稳定的细胞系 前病毒基因组将被分离。肿瘤坏死因子-α或PMA的作用将是 不同来源肝癌细胞感染性克隆的鉴定 台词。6)蛋白激酶C和cAMP在肿瘤坏死因子-α中的作用 对PMA效应进行了研究。7)凝胶滞留分析和广泛的 DNA足迹将用于识别肿瘤坏死因子-α或 结合HIV-1和TAR调控区的PMA处理细胞 RNA序列。8)通过突变这个前病毒克隆的不同区域，我们 打算将HIV-1基因组中负责肿瘤坏死因子的区域(S)本地化。 α或PMA介导的抑制作用。9)我们将识别、描述、 提纯，如果需要，分离编码反式作用的cDNA 多种肝癌和其他细胞系中与HIV-1 LTR结合的蛋白质 通过筛选lambdagt11文库并与处理细胞进行比较。

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.