CELLULAR GENES REQUIRED FOR HIV-1 INFECTION AS TARGETS FOR ANTI-VIRAL THERAPY

(1)项目目标： - 确定最佳HIV-1感染所需的细胞基因，或 参与HIV-1发病机制。 - 研究胸腺内HIV-1感染影响免疫功能的机制， 不同T细胞亚群的发育。 - 为了了解HIV-1感染如何导致高水平的编程性 CD 4和CD 8细胞。 - 将获得的知识应用于抗病毒治疗的开发 针对病毒和细胞基因。 (2)实验方法： - 一组突变体来源于人T细胞系克隆CEM， 显示出对HIV-1感染的不同易感性。多 开发了分析这些克隆的测定法。 - Hu/Scid小鼠胸腺内感染原代分离株， 艾滋病。胸腺细胞经3种颜色染色后可分为不同的亚群 并通过定量PCR确定前病毒DNA的存在。 - 负性激酶lsk在Fas介导的细胞凋亡中的作用是 使用一组缺乏LSK或表达LSK的人细胞系测定 野生型或突变的LSK。 (3)主要发现： - 分离出两个CEM亚克隆，其具有显著降低的 易受多种HIV-1病毒株感染。这些突变体 发现DNA结合蛋白水平显著降低 属于NFk-B家族（p50）。NF-kB蛋白水平降低 不影响细胞生长，仅影响HIV-1易感性。这些基因可以 作为反义抑制的靶点，以维持患者的PBL在 静止状态，防止整合病毒的再激活。 出版物： J. Qian，V. Bours，J. Manischewitz，and H.戈尔丁 1994. J. Immunol. 152：4183-4191。 - Hu/Scid模型表明，在感染的胸腺中， 成熟细胞也被病毒感染。它可能发生在 CD 4 + CD 8+感染细胞发展为单阳性CD 4和CD 8 细胞 - 发现LSK激酶对 Fas介导的凋亡。据推测，在艾滋病毒感染者中， 细胞内LSK可能减少，导致增强 细胞凋亡的易感性。

(1) Goals of project: - To identify cellular genes required for optimal HIV-1 infection, or involved in HIV-1 pathogenesis. - To study the mechanism by which intrathymic HIV-1 infection affects the development of different T cell subsets. - To understand how HIV-1 infection leads to high levels of programmed cell in both CD4 and CD8 cells. - To apply the knowledge gained towards development of anti-viral therapy which target both viral and cellular genes. (2) Experimental approach: - A panel of mutants were derived from the human T cell line clone CEM, demonstrating different susceptibilities to HIV-1 infection. Multiple assays were developed to analyze these clones. - Hu/Scid mice were infected intrathymically with primary isolate of HIV. Thymocytes were separated into various subsets by 3 color stainings and the presence of proviral DNA was determined by quantitative PCR. -The role of the negative kinase lsk in fas-mediated apoptosis was determined using a panel of human cell lines lacking lsk or expressing wild type or mutated lsk. (3) Major Findings: - Two CEM subclones were isolated with dramatically reduced susceptibility to infection by multiple strains of HIV-1. These mutants were found to have a markedly reduced level of DNA binding protein belonging to the NFk-B family (p50). The reduced levels of NF-kB proteins does not effect cell growth only HIV-1 susceptibility. These genes could be the target of anti-sense suppression to maintain patients' PBL at a resting state, preventing reactivation of integrated virus. Publication: J. Qian, V. Bours, J. Manischewitz, and H. Golding. 1994. J. Immunol. 152: 4183-4191. - The Hu/Scid model demonstrated that in the infected thymus, CD8+ mature cells are also infected with the virus. It may occur during the development of CD4+CD8+ infected cell into single positive CD4 and CD8 cells. - The lsk kinase was found to have strong inhibitory effect on FAS-mediated apoptosis. It is postuLated that in HIV-infected patients the intracellular of lsk may be reduced, leading to enhanced susceptibility to apoptosis.